When a growth factor binds to the plasma membrane of a quiescent cell, an intracellular signaling pathway is activated telling the cell to begin growing. A key molecule in this signaling pathway is the GTP-binding protein, or G-protein, Ras. Ras can act as an on-off switch telling the cell to grow or not. In its inactive form, Ras is bound to GDP while in its active form it is bound to GTP. This exchange of nucleotides is catalysed by guanine nucleotide-exchange-factors (GEFs). The return to the inactive state occurs through the GTPase reaction, which is accelerated by GTPase-activating proteins (GAPs). In Part 1 of his talk, Dr. Wittinghofer explains how solving the three-dimensional structure of Ras, and other G-proteins, allowed him to understand the conserved mechanism by which G-proteins can act as switches. The structure also identified domains unique to each G-protein that provide the specificity for downstream signals.
In the second part of Dr. Wittinghofer’s talk he explains the link between GTPases and disease. Ras is both a key molecule in regulating normal cell growth and an oncogene in unregulated cancer cell growth. Mutations in Ras that prevent the hydrolysis of GTP to GDP lock Ras into an active state rendering it independent of upstream growth factor signals. Biophysical studies from Wittinghofer’s lab solved the multiple steps in the hydrolysis of GTP to GDP and explained why particular mutations in either Ras or Ras-GAPs cause unregulated activation of Ras and tumor formation. Examples of other G-proteins that are unable to hydrolyse GTP and result in different diseases such as Retinitis Pigmentosa, are also presented.
All Course Materials for this Session (Educators only) – Created by Alfred Wittinghofer
00:00:01.14 Hello, my name is Alfred or Fred Wittinghofer and I'm an emeritus group leader
00:00:07.24 from the Max-Planck Institute for Molecular Physiology in Dortmund, Germany.
00:00:13.06 What I would like to tell you today is about a class of proteins
00:00:18.08 that bind GTP (so GTP-binding proteins) that work as molecular switches.
00:00:23.11 And I will tell you in my first lecture, how they work
00:00:26.14 and in the second lecture, how they lead to a number of different diseases that we have studied.
00:00:32.29 So imagine, for example, that you have a quiescent cell that sits there in the G0 phase.
00:00:38.04 It doesn't grow, doesn't differentiate and it needs a signal from the outside
00:00:43.04 in order to start proliferation right away.
00:00:45.14 And the way it works is that a growth factor hits the cell
00:00:48.22 and induces a series of reactions indicated here by these arrows.
00:00:53.14 And then this series of reactions comes to the cell nucleus
00:00:57.10 where DNA is duplicated and the cell decides to proliferate.
00:01:00.09 And one of the most important elements in the signal transduction chain is Ras
00:01:06.10 a GTP-binding protein, one of the leading molecules in this class
00:01:12.09 and this then regulates cell growth as a molecular switch.
00:01:17.21 And you can imagine, if this regulation does not work,
00:01:21.21 and cell growth is uncontrolled, then you have cancer.
00:01:25.26 And Ras is one important element of cancer formation
00:01:28.16 and I will talk about that in my second seminar.
00:01:33.18 In another example, for example, you have a quiescent cell
00:01:37.04 where you have the actin cytoskeleton marked here in the left part of the pictures
00:01:44.16 where the actin cytoskeleton is very diffuse in blocks and small stripes and so on
00:01:51.16 and then suddenly you hit these cells with
00:01:53.15 a particular class of GTP-binding proteins called Rho
00:01:56.15 and then you see what happens.
00:01:58.05 In one case you get stress fibers,
00:02:00.14 in the other case you get a structure at the cell periphery which are called lamelopodia
00:02:06.26 or here you get structures that are making long extensions which are called philopodia
00:02:13.00 which make the cell move or proliferate or differentiate and so on and so on.
00:02:18.21 And these reactions are also controlled by a GTP-binding protein
00:02:22.27 and they are called Rho, Rac or Cdc42.
00:02:26.13 So the question then boils down to the thing,
00:02:29.28 how do you construct a molecular switch that is reversible,
00:02:33.17 that can be regulated at any level and then does the thing that it's supposed to do?
00:02:40.19 And so nature has devised a very large class of proteins called GTP-binding proteins
00:02:47.03 that comes in two flavors; in the GTP bound state it's on and in the GDP bound state it's off.
00:02:53.01 So the difference between these two states is a single phosphate.
00:02:56.08 And to show that this is a very important class of proteins,
00:03:02.29 we can find more that 38,000 GTP-binding proteins or G proteins, as I would like to call them,
00:03:08.29 in about 1300 genomes by December 2010.
00:03:13.20 So that tells you these are really important molecules found in all kingdoms of life
00:03:18.21 and some of these proteins are the most highly conserved proteins in nature at all.
00:03:24.25 So, let me tell you about how these molecular switches work.
00:03:31.05 And I will talking mostly about Ras-like proteins because these are the ones that we work with
00:03:35.22 and they are sort of the prototype for learning how these are regulated.
00:03:40.01 So you start out with a signal that comes, for example, like a growth factor
00:03:44.29 or whatever, that hits, somehow these G proteins, (and I will be talking to you about that later)
00:03:50.16 and that induces a series of steps that lead to the protein becoming loaded with GTP.
00:03:58.01 And then it has its downstream effect.
00:04:00.10 And in Ras-like proteins it works the following way:
00:04:04.29 these nucleotides are usually bound very tightly (picomolar range)
00:04:09.27 such that GDP never comes off by itself
00:04:12.15 but needs the action of a nucleotide exchange factor which is called GEF (guanine nucleotide exchange factor)
00:04:19.12 which allows GDP to be released much faster
00:04:25.16 and then allows GTP to bind to the protein.
00:04:27.25 And now it is active.
00:04:29.16 And now it can do its effect but you obviously, since it's a molecular switch, you want it to be switched off again.
00:04:36.06 And the way you do that is not the reverse, not the exchange of GTP for GDP,
00:04:42.10 but rather it is the irreversible step, the GTPase hydrolysis.
00:04:48.02 So GTP is hydrolyzed to GDP and Pi and there is another protein that
00:04:53.20 stimulates that reaction because it is intrinsically very slow
00:04:57.27 and becomes stimulated by a protein called GTPase Activating Protein or GAP.
00:05:04.07 And I will be talking about that, obviously, in great detail in my second seminar
00:05:08.07 because that is where you see a lot of diseases being due to inability to hydrolyze GTP.
00:05:15.28 So the downstream effector is then something that is mediating the biological effect
00:05:22.10 and the effector is a molecule that recognizes, specifically, only the active
00:05:26.10 GTP-bound form and not the inactive GDP-bound form.
00:05:31.21 So, just to make you familiar with the way this thing can work,
00:05:36.14 since the cycle of GDP to GTP is regulated by Kdiss or Kd (dissociation) for GDP
00:05:49.19 or is regulated by the GTPase reaction, which is Kcat or Koff,
00:05:55.18 you can see that the signal can either increase Kdiss or it can decrease Koff.
00:06:02.10 In both cases you get an increase in the effect, in the biological effect
00:06:07.18 because the effect is finally determined, really, by the GEF reaction or the GAP reaction.
00:06:16.08 And you can quantify this and say that the amount of biological effect
00:06:21.13 that is coming out of this system is
00:06:23.27 directly proportional to Kon (to the introduction of GTP)
00:06:28.25 or is inversely proportional to Koff (to the GTPase reaction).
00:06:33.26 If you make Kdiss faster, you get more GTP bound to protein
00:06:38.13 or if you make the GAP reaction slower you also have an increase in GTP.
00:06:45.18 So, let's now come to how these proteins look, how you recognize them and so on.
00:06:51.10 So, obviously, are there sequence motifs?
00:06:53.14 Are there structures or biochemistry--are they similar between these proteins?
00:06:57.23 Yes, indeed. What I will show you is that you can identify
00:07:01.17 these proteins very easily from sequence motifs, from structure
00:07:05.10 and also the biochemistry are rather similar between all these different proteins.
00:07:09.25 And you can present some of the general rules for recognizing
00:07:14.13 and working with these proteins which I will do in the next 30 minutes.
00:07:21.01 So there are, obviously, when you look at a new protein you may have sequenced
00:07:27.10 and then you compare the amino acid sequence,
00:07:30.00 if you find these 5 sequence elements, indicated here,
00:07:33.01 called G1, G2, G3, G4 and G5, standing for G binding motifs,
00:07:40.24 then you immediately know that you're dealing with a GTP binding protein or G protein.
00:07:45.22 And these elements are, for example, the first one is the so called P loop
00:07:51.04 is a motif G 4-times x (which means any amino acid there)
00:07:56.28 then another conserved glycine, a conserved lysine, S or T,
00:08:02.25 and this is one of the most frequently occurring sequence motifs in the database
00:08:09.03 because not just G binding proteins but also ATP binding proteins have this sequence motif.
00:08:16.00 The second one is just the conserved threonine
00:08:18.22 and a conserved D x x G as the Switch I and Switch II motifs.
00:08:23.11 I will show you later on what Switch I and Switch II means.
00:08:26.00 And lastly there are two motifs, N K x D (G4) or s A k
00:08:33.20 where only, for example in the last motif, the alanine is totally conserved.
00:08:38.19 These are the motifs that are involved in binding the nucleotide
00:08:41.23 and also are involved in the specificity of the nucleotide.
00:08:47.07 And I should also remind you that there are a few proteins
00:08:51.15 which are also GTP-binding but they have a different fold and a different sequence and so on.
00:08:57.16 These are the most famous examples: tubulin or the bacterial homolog FtsZ
00:09:02.24 which you have heard about in other of these iBio seminars for example from Ron Vale.
00:09:08.14 And there are also a few metabolic enzymes.
00:09:11.23 So it's a strange coincidence that almost all the metabolic enzymes
00:09:17.07 that need energy to catalyze the chemical reaction, that they use ATP and not GTP
00:09:24.15 and only very few examples, indicated here, use GTP.
00:09:28.00 So that seems to be as if nature decided that in order to transmit energy it uses ATP
00:09:34.01 and for the regulation of processes it uses GTP,
00:09:38.14 except for these few examples here.
00:09:44.00 So obviously as biochemists we...or lets say a structural biochemist,
00:09:48.18 that I and my lab is involved in, we would like to
00:09:52.28 understand the system we're working with and the biochemistry and the biology of it
00:09:56.22 by knowing the structure because this is usually giving you the most deep understanding of your favorite system.
00:10:08.05 So, to be brief, for those of you that have never worked with protein structures...
00:10:13.10 and we're using x-ray crystallography to determine the structure
00:10:16.05 although there are other methods that I will not be talking about
00:10:19.01 and there will be actually an iBio seminar series coming up later on.
00:10:24.05 But, I'll give you a brief introduction
00:10:27.10 to give you a feeling for how you get at your particular structure.
00:10:30.21 So you can see that you're not afraid of using the method in your favorite system.
00:10:36.13 So what you start out with is you have to have pure protein
00:10:40.19 sometimes a lot or, let's say lots of milligrams of protein.
00:10:44.23 You crystallize them. Hopefully that works.
00:10:48.07 And once you crystallize them you put them through an x-ray beam.
00:10:51.26 And sorry if this is still a German slide
00:10:56.02 because Mr. Rontgen is the one who discovered x-rays
00:11:00.09 and we still like to call them Rontgen-strahlung which means x-rays.
00:11:05.10 And so you shine these x-rays through a crystal and then these x-rays are diffracted.
00:11:11.22 You sample the diffraction pattern and then you use that to calculate your result.
00:11:19.22 Just to make sure you understand this slide I put the English version of it down there.
00:11:25.09 X-rays shone into a crystal and then being analyzed on a detector.
00:11:30.13 So you end up, by a complicated mathematical calculation,
00:11:35.12 which is standardized by the way so you don't have to really learn all the details about it,
00:11:40.07 but under that method you end up with an electron density map of your favorite protein
00:11:45.09 and now you have to do the really fun part which is build an atomic model of it.
00:11:51.00 And I give you here an example of a particular part of Ras polypeptide. So the chain runs from left to right.
00:11:57.15 You see that, for example, this would be a 5-membered ring that can only be proline.
00:12:06.09 You see, for example, down there, a bifurcated amino acid
00:12:10.00 which can be aspartic or threonine or valine.
00:12:12.07 The same one down here.
00:12:13.18 And there's an aromatic residue up there which has also a little tip on it so that must by a tyrosine.
00:12:19.22 So in other words, you end up
00:12:22.00 (and you probably did it while you were looking at the screen)
00:12:25.21 you end up with your atomic model with the tyrosine, then an aspartic acid,
00:12:31.03 a proline, and a threonine down there
00:12:33.01 and if you look into the sequence of your particular protein
00:12:35.16 you know this must be a certain part of the sequence of your protein.
00:12:41.10 So you end up then, in the end, with a ribbon model of your complete protein.
00:12:47.07 And you can see here that this one is composed of alpha helices and beta sheets
00:12:52.04 and that's why I just call it an alpha-beta protein
00:12:55.14 which is, by the way, typical for any nucleotide binding proteins
00:12:59.01 which is an alpha-beta fold.
00:13:01.14 If you go into the database you will see that.
00:13:04.09 So, if you now look at the sequence motif that I have indicated to you a while ago,
00:13:11.17 G1 to G5, you can now see...where can I find them?
00:13:17.00 And they're actually found only in the loops.
00:13:19.14 So, for example, if you see the first beta strands...
00:13:22.10 so this is the N-terminus, the first beta strand, you go through G1 into the other helix down here
00:13:27.28 and then down into the other loops down here
00:13:31.12 and you see that G1, G2, G3, G4 are all in loops
00:13:35.18 which is again very typical for a protein structure--that the actual fold is a very stable entity
00:13:43.01 and the business part of the protein where you see changes,
00:13:46.29 where things bind and are released,
00:13:50.06 where things are hydrolyzed or chemical conversions are happening,
00:13:54.02 they are happening in loops that combine these structural elements.
00:14:00.00 And if you look at the structure you see, actually, that
00:14:02.22 all the conserved elements here are really on just the one part of the structure right here.
00:14:11.07 And the other part, down there, is probably unimportant
00:14:14.19 in terms of, at least, the interactions with other molecules.
00:14:19.05 Just to give you a flavor of some of the motifs that we are dealing with here,
00:14:24.20 this is, for example, the P loop
00:14:26.25 which is a connection between the beta strand down there, it goes though a loop and ends up in the helix.
00:14:33.27 And you see that the first conserved glycine (which has the number in Ras by the way)
00:14:39.24 and going through the loop. Coming to the other conserved glycine on top, there.
00:14:44.02 The conserved lysine is here and then a serenine or threonine.
00:14:47.23 So what you actually see is that the phosphate sits right in the middle of this loop
00:14:52.29 and that's why it is some how neutralized by the charges in the loop
00:14:59.04 and this the most frequent sequence motif in the database.
00:15:05.02 Many ATP-binding and GTP-binding proteins just contain this motif.
00:15:10.00 For example, you heard about kinesin, about myosin in these iBio structure series,
00:15:14.15 and they also contain the same type of motif.
00:15:20.00 This is shown here again in a little more detail.
00:15:23.15 You can see that the beta sheet that sits right in the middle of this P loop
00:15:28.13 makes the main chain interactions and lysine interaction
00:15:33.02 so that the negative charge of phosphate is neutralized by binding into the P loop.
00:15:39.04 It is also called the polyanion hole
00:15:41.10 by Georg Schulz many many years ago
00:15:43.07 when he worked adenylate kinase.
00:15:47.19 So, now we know the structure of Ras and that was the first structure to be solved
00:15:53.29 many years ago by us.
00:15:56.04 So in order to look at different structures
00:15:59.26 let me first introduce you to the Ras super family of GTP binding proteins
00:16:03.08 where each of the sub-families is, first of all, defined by sequence.
00:16:09.14 So proteins within the sub-family are more similar to each other than proteins outside the sub-families.
00:16:14.09 And if you align them by sequence you also align them by function because
00:16:19.10 each sub-family is involved in some kind of different function.
00:16:23.03 For example, the Ras sub-family is involved in general signal transduction reactions.
00:16:28.03 The Rab family is involved in vesicular transport.
00:16:31.26 The Rho protein sub-family, I introduced you to already, regulates the actin cytoskeleton.
00:16:37.11 And I showed you Rho, Rac and Cdc42.
00:16:40.10 And for example, the Ran sub-family is involved in nuclear transport.
00:16:45.04 It's a nuclear version of Ras. That's why it's called Ran.
00:16:47.22 And Rho, for example, is called Rho for Ras Homology.
00:16:51.05 Rab is called Rab for Ras in the brain and so on.
00:16:54.22 So all the names derive, actually, from the grandfather of the family, which is Ras.
00:17:00.00 So we have been working with a number of these proteins
00:17:02.20 and I will show you a few examples of these
00:17:05.03 and just, for example, compare their structure.
00:17:07.25 And we'll also talk about function in my second seminar.
00:17:12.04 So that indicates that a number of structures have been solved
00:17:16.07 in the meantime by us and many other people.
00:17:18.12 And the first correct structure of Ras was in 1989
00:17:23.13 and we now have about 400-500 deposits in the pdb database.
00:17:29.24 And not just the protein itself, but also complexes with effectors;
00:17:34.00 complexes with GEFs and complexes with GAPs.
00:17:37.02 Obviously, these helped us to understand, actually, the complicated regulation of these proteins
00:17:41.14 much better than only biochemistry would have done.
00:17:46.03 So if you look now, let's say, at a few of these structures,
00:17:48.21 on a first view you would say, immediately, "Yeah, they look totally identical."
00:17:53.11 which is obviously true. If you, for example, compare Ras and Rap, they are rather similar
00:17:59.10 and also Rho and Cdc42 and also Rab.
00:18:06.00 So the overall fold is the same but you see small additional elements.
00:18:09.22 For example, in the Rab-Rho family you see this extra helix here and there.
00:18:15.04 And in the Arl family you see an extra N-terminal helix.
00:18:20.00 But other than that, you see that the structure is the same.
00:18:22.11 So you would ask yourself, "Why would these proteins do different things if they have the same structure?"
00:18:26.26 And obviously the answer is very simple.
00:18:30.05 It's not the fold that determines what these proteins are doing in the biological system.
00:18:34.10 It's what they're interacting with
00:18:36.16 and the interaction is determined by the surface of the protein where you have different amino acids.
00:18:41.18 This can be indicated, for example, by just looking at the charges of the surface
00:18:46.15 where red means negative charge and blue means positive charge.
00:18:50.17 And now if you compare, for example, the similar Ras and Rab proteins you see, really,
00:18:55.25 at the surface it's different enough to
00:18:58.11 make sure that the Ras protein interacts only with its downstream effectors
00:19:02.26 and Rap interacts only with its downstream effectors.
00:19:06.05 And the same is true for the Rho protein which is different from Cdc42
00:19:11.12 but is also different from Ras and Rap and so on and also Arl and so on.
00:19:15.25 So the message from all of this is that the overall fold is exactly the same for
00:19:21.05 all of these proteins but they have additional elements and they have a different surface
00:19:25.01 that makes these proteins do its particular biological reaction that they are involved in.
00:19:32.13 So, the next thing that I would like to introduce you to is how the switch works.
00:19:38.22 So. obviously, in our schemes I have also always shown that the GDP-bound form
00:19:43.25 is different from the GTP-bound form by a symbol.
00:19:47.01 But now we will obviously look at the structure and biochemistry
00:19:49.21 and would like to know: How does this structure actually change
00:19:52.05 when the protein goes form the inactive to the active conformation.
00:19:56.02 So the basic element of the switch mechanism that we would like to understand...
00:20:02.12 how does the protein change structure when it goes from the GDP-bound to the GTP-bound state?
00:20:07.17 ...is the following...most of the structure (which is this grey part down here)
00:20:14.25 does not change at all when the protein goes from one state to the other.
00:20:18.18 But there are two elements in the structure called switch I, down there, and switch II, down here
00:20:24.13 which is part of the conserved elements G2 and G3.
00:20:27.20 And they contain two amino acids; threonine-35 is the number in Ras
00:20:35.24 and glycine-60, again the number in Ras
00:20:38.00 and in different proteins the number would be different.
00:20:40.08 They are bound to the gamma phosphate by these two main-chain hydrogen bonds
00:20:44.05 which are indicated here as springs that are loaded by binding to the gamma phosphate.
00:20:49.13 So switch I and switch II are really important elements of the structure change
00:20:55.22 and they are obviously called switch I and switch II because
00:20:58.23 they change their structure when they go from GDP-bound to GTP-bound.
00:21:02.04 These are the important elements for the biological function because
00:21:07.02 that's where they change structure when going from one form to the other.
00:21:12.07 And I will show you a number of examples of how this looks in detail.
00:21:16.21 For example, in the case of Ras, you see that if you overlay
00:21:21.09 the structure of GDP- and GTP-bound conformations they are almost totally identical
00:21:26.18 in most of the secondary structure elements, shown as beta sheets and helices here.
00:21:32.01 And the only changes, really, are happening in the switch regions,
00:21:35.03 which are down here in this colored area here.
00:21:38.15 where, for example, you have a tyrosine-32 that sits inside and goes outside
00:21:44.16 and you have a threonine down here which goes from the outside to the inside.
00:21:48.20 So you see these two different changes in switch I up there (which is the purple color)
00:21:54.27 and the cyan color down here (which is switch II) you see, again, structural changes,
00:21:59.06 a melting of the helix down there.
00:22:01.02 So there's a localized change
00:22:03.06 and this would be the part where, obviously, proteins that
00:22:06.18 are acting downstream of Ras would be recognizing this part of the structure
00:22:12.07 which is shown here again in a movie.
00:22:16.00 You'll see the grey part of the structure doesn't change much.
00:22:18.07 What is happening is down there.
00:22:20.07 You see these colored loops that are changing
00:22:23.16 and this is element where proteins that would recognize this Ras in the GTP-bound conformation
00:22:29.09 would come and recognize this conformation.
00:22:31.18 We can show the same thing by coloring the surface of the protein
00:22:36.12 and you see here in color again the two switches when they go from GDP- to GTP-bound conformations.
00:22:43.17 And this is actually a simulation of the reaction between the two states.
00:22:48.08 But what you see again, is that the surface of the protein
00:22:51.01 where the action happens changes when it goes from one state to the other.
00:22:57.02 And so that's where the business end of the protein is.
00:22:59.15 This is where almost all the factors that recognize Ras would attack the protein.
00:23:04.20 Just to give you a more dramatic example:
00:23:08.11 So the conformation change is canonical. I showed you that in one of the previous pictures.
00:23:13.08 But there are some wonderful dramatic changes that we can observe
00:23:17.14 by looking at different Ras-like proteins,
00:23:19.28 for example, the protein Ran, that I introduced you to briefly before,
00:23:23.23 a protein that regulates nuclear transport
00:23:26.29 and has a the C-terminus an extra element, an extra helix, which we call the C-terminal helix
00:23:33.10 which is bound by its highly negatively charged end to the positively charged protein.
00:23:39.20 So it sits on the surface in the GDP-bound state.
00:23:42.12 Now, see what happens in the GTP-bound state.
00:23:45.06 You see that now you have the canonical triggering of the switch
00:23:50.16 down at the gamma phosphate side which would be somewhere here.
00:23:53.05 The two switches no change their structure and by doing that
00:23:56.28 they kick out the C-terminal end which does something involved in nuclear transport.
00:24:04.15 An even more dramatic example is shown by the protein Arl or Arf.
00:24:10.02 So here you see a protein up in the GDP-bound state.
00:24:14.06 You see there's alpha and beta phosphate bound but the gamma phosphate
00:24:19.01 binding site is very far away from where it should be if its in the canonical structure.
00:24:24.12 So what happens now, if you have a tri-phosphate bound to the protein down there,
00:24:28.25 it moves what is called the inter-switch region which are actually two beta strands.
00:24:33.22 It moves it by two amino acids towards the N-terminus
00:24:36.21 and kicks out the N-terminus and thereby induces a large conformational change
00:24:41.23 which is shown in a movie again, down here.
00:24:45.01 So you see these two beta strands that move by a very large distance
00:24:49.07 and kick out the N-terminal end,
00:24:51.16 which, the N-terminal end is actually interacting now with the plasma membrane
00:24:54.28 and does something that has to do with vesicular transport.
00:24:59.07 So the message from all of these things is that there is a canonical change
00:25:05.21 when the protein goes from GDP-bound to GTP-bound.
00:25:10.19 The trigger is the same but the affects can sometimes be very dramatic.
00:25:15.10 And I showed you another dramatic example for that.
00:25:20.01 And, incidentally, you have heard a lot in different seminar of this series
00:25:28.26 that motor proteins like myosin or kinesin also do a conformational change
00:25:33.06 between the ATP- and ADP-bound state.
00:25:35.20 And it turns out that the mechanism for that structural change is
00:25:40.07 almost totally identical to that of G protein.
00:25:42.20 So you see here, for example, in red on the left side, for the motor protein,
00:25:46.23 the conserved sequence element.
00:25:49.00 And on the right you see the sequence elements that I have introduced you to before
00:25:53.03 which from GTP-binding proteins and they are actually very very similar.
00:25:56.17 And the conformational change is very similar,
00:25:59.19 except that in the case of motor proteins it is transmitted to do work.
00:26:04.04 So, for example, in myosin the conformational change of the switch II
00:26:07.15 makes a lever arm movement that makes the protein walk along the actin filament.
00:26:12.10 And in the case of kinesin it walks along the tubulin filament.
00:26:18.02 And so in Ras this conformational change is obviously converted not into work
00:26:22.23 but is used to make an irreversible change from GTP-bound to GDP-bound state
00:26:28.15 by the GTPase reaction.
00:26:31.16 So let me at the end, or towards the end,
00:26:35.18 show you some of the biochemical properties of the protein,
00:26:39.07 in particular, of small G proteins which we usually work with.
00:26:42.27 So all of them have a high affinity to nucleotides.
00:26:47.26 High affinity means in the pico molar or nano molar range.
00:26:51.01 Which means that (and this is the second point here)...
00:26:55.07 that means that the dissociation of nucleotide is very slow.
00:26:58.17 The half-life of dissociation at 37 degrees for GDP would be on the order of 20 minutes to 30 minutes.
00:27:06.00 Which would be way too long for a signal transduction process
00:27:10.18 where this protein is activated within minutes.
00:27:13.04 And that's why, obviously, you have these nucleotide exchange factors
00:27:16.07 that I showed you before.
00:27:17.17 The third thing that I would like to introduce you to is that the affinity is magnesium dependent.
00:27:23.20 So in the absence of magnesium, for example, the affinity
00:27:27.06 of nucleotide is 1000-fold less which is a very interesting technical thing for us.
00:27:32.25 I will show you that in a minute also...so, why we can work with these proteins.
00:27:37.02 You can reduce the affinity just by removing magnesium.
00:27:40.19 The fourth things that's here on the slide is
00:27:43.23 that there is a very high specificity for guanine nucleotide.
00:27:47.17 For example, I told you the affinity is in the order of pico molar to nano molar.
00:27:52.02 But the affinity to ATP or adenine nucleotide is in the order of millimolar.
00:27:58.21 So, a huge difference in affinity of guanine nucleotide versus adenine nucleotide.
00:28:05.10 And I'll, again, show you that in a minute, how that comes about.
00:28:07.25 Also, I showed you already that the GTPase is very often very slow,
00:28:13.06 again, with a half-life at 37 degrees of 20 to 30 minutes.
00:28:17.27 And again, in a biological process, you want this reaction to be much faster.
00:28:21.27 So, actually, almost all G proteins are very slow enzymes, very slow phospho-transfer enzymes.
00:28:29.12 But, they become, obviously, very good transfer enzymes
00:28:32.25 when in the presence of the GTPase Activating Protein.
00:28:36.08 And finally, again, which is a very universal observation, the GTPase reaction
00:28:45.05 just like the ATPase reactions, are always magnesium dependent.
00:28:49.00 And I'll show you again what that looks like.
00:28:50.20 So, you need a divalent ion first to make it high affinity
00:28:54.29 and for the GTPase reaction.
00:29:00.06 So for example, how does the specificity of guanine nucleotide come about?
00:29:05.02 Here you see the two elements that I introduced at the very beginning,
00:29:08.03 G4 and G5 down there which is N or T KxD which is the G4 motif
00:29:16.15 or the sAk where alanine is probably the only really conserved element.
00:29:22.12 And what you see is the guanine base making a very strong, bifurcated hydrogen bond
00:29:27.21 to this aspartic acid down there which obviously adenine couldn't do.
00:29:32.27 And also the alanine makes a main chain hydrogen bond with the oxygen
00:29:37.24 which would be the amino group in the case of adenine and again that couldn't happen.
00:29:41.21 And you see that the lysine which sits underneath the base
00:29:44.18 and the alanine make additional contacts.
00:29:47.04 So that's the reason for the specificity.
00:29:50.15 And also if you mutate any of the residues around the guanine base
00:29:55.13 binding site you make the binding very much lower affinity.
00:30:01.23 So here you see the essential magnesium ion.
00:30:07.06 It is bound between the beta and the gamma phosphate.
00:30:11.06 These are two of the ligands of magnesium.
00:30:13.17 You see there are two ligands coming from the protein itself; a threonine and a serine.
00:30:18.10 This is always conserved in the GTP-bound state for all GTP-binding protein.
00:30:22.22 And then you have two water molecules that make the coordination field complete.
00:30:30.17 And a general theme of any ATPase and any GTPase is that you have magnesium as
00:30:37.22 a bidentate interaction like this one here where the gamma phosphate is to be transferred
00:30:43.12 but if you have a cleavage at the alpha phosphate here,
00:30:46.14 magnesium is sitting between alpha and beta.
00:30:48.10 So, that's a general thing:
00:30:50.15 that you need a bivalent ion to neutralize the charges of the phosphate.
00:30:57.26 And I will talking about that, obviously, in my second seminar
00:31:00.23 where we'll talk in detail about the GTPase reaction.
00:31:05.01 Another thing that is very important for analyzing and working biochemically with
00:31:11.15 these proteins is the high affinity is such that if you isolate the protein, let's say
00:31:18.03 from E. coli (make it recombinately), that you want to make sure that
00:31:23.03 you know what nucleotide is bound and to what extent.
00:31:28.03 So, if you run, for example the protein, on an HPLC column,
00:31:31.06 you see this peak here at a certain elution volume.
00:31:34.19 If you compare that to a standardized elution diagram
00:31:39.20 where you take a mixture of GMP, GDP and GTP in a control reaction you see that this is GDP.
00:31:47.05 And you can also quantify that because this has been equilibrated
00:31:52.23 with a certain about of nucleotide such that you now know
00:31:55.28 that your protein has a 1:1 complex of protein and GDP,
00:32:00.26 which is important because if you want to analyze the protein
00:32:04.07 in any of the reactions that this protein does
00:32:06.12 you want to make sure that you know which protein is bound to it already and to what extent.
00:32:13.15 People make a lot of mistakes by forgetting that the protein already has nucleotide
00:32:19.24 and that is important if you analyze the following reaction that I will show you.
00:32:25.12 For example, you want to do an exchange.
00:32:29.12 You need to to convert the protein into the form that
00:32:34.03 you would like it to be in in oder to analyze the following reaction.
00:32:37.24 And the way you do that is you use EDTA to make a very quick exchange of nucleotides.
00:32:44.09 So, EDTA picks off the magnesium
00:32:46.04 and without EDTA you see here that the exchange reaction would be very slow.
00:32:49.16 So you're looking at the reaction of Ras-GDP and you want to introduce a GTP version of that,
00:32:55.17 for example, tritium labeled GTP or gamma-P32 labeled GTP.
00:32:59.20 Here, you see that reaction would take a very, very long time in order to get to completion.
00:33:04.19 But in the presence of EDTA, it takes minutes
00:33:06.27 and you have a full conversion of the protein into the GTP-bound form, for example,
00:33:11.16 if you want to, let's say, analyze the GTPase reaction
00:33:14.17 because if you start with the GDP form,
00:33:17.24 the rate-limiting step would be exchange, rather than the GTPase reaction.
00:33:22.15 And we also use, for example, of fluorescent analogs of GDP, which is shown here.
00:33:31.15 So you have on the ribose or the deoxyribose (depending on which you want to use)
00:33:36.06 you have a fluorescent reporter group which is called mant or mGDP or GTP
00:33:42.12 which is a very sensitive probe for the interaction of the protein with
00:33:47.18 other proteins, with nucleotide itself and so on.
00:33:50.14 And we have shown that this fluorescent reporter group does not
00:33:54.05 disturb most of the reactions of the protein that we are working with.
00:34:00.03 For example, here, this shows you what a wonderful fluorescent reporter group this is.
00:34:05.13 So, the lower curve here would be the emission curve of the mant-GDP or -GTP
00:34:13.18 which is a certain amount of emission spectrum you get
00:34:17.16 and if you add Ras you see that you get a huge increase in the fluorescence emission
00:34:25.04 which means, probably, that the probe is now in a completely different environment
00:34:29.00 and that's why you get such a wonderful change
00:34:31.18 which you can use now to do lots and lots of different reactions.
00:34:34.29 Just to again give you an example, you first load the protein with a fluorescent analog
00:34:40.26 (this is Ras bound mGDP) and now you want to analyze the exchange reaction
00:34:45.22 with one of the guanine nucleotide exchange factors that I showed you in the beginning.
00:34:51.28 So, in the absence of an exchange factor, at room temperature,
00:34:57.19 there would be almost no exchange in the time frame that is shown here (600 seconds).
00:35:03.10 But now, if you add one of the exchange factors
00:35:05.22 (which is obviously the catalytic domains of one of the exchange factors),
00:35:08.27 you see you get a very fast release of nucleotide.
00:35:13.05 The way we do this is we take a large excess of unlabeled GDP
00:35:17.10 to replace the fluorescent derivative and thereby, you get a decrease in fluorescence
00:35:25.01 because when the mant-GDP becomes free, fluorescence is about one half of what it was before.
00:35:31.02 And you use a very high excess of GDP in order to
00:35:35.05 make the back reaction spectroscopically silent.
00:35:38.18 In other words, you can now analyze this by a first order reaction analysis
00:35:45.10 and say what is Kcat of the exchange reaction of Ras + Sos.
00:35:51.11 In the very end, let me briefly show you some multi-domain G-proteins
00:35:59.18 because so far I have talked, mostly, about Ras-like proteins which consist mostly, of just the G domain.
00:36:05.13 And obviously there are a larger number of G proteins that consist of
00:36:10.14 many more domains and are much, much larger.
00:36:13.04 So the proteins that we talked about, the Ras-like proteins are in the order of 20 kDa
00:36:17.14 but the proteins that are indicated here are much, much larger.
00:36:21.17 For example, the translation factor...
00:36:24.17 so these are the factors that regulate ribosomal biosynthesis
00:36:28.04 which are called EF-Tu or EF-1, EF-G and so on.
00:36:32.12 So all of these use GTP conversion
00:36:36.10 for driving some part of the ribosomal synthesis and these are between 40 and 80 kDa large.
00:36:44.11 And these are the most highly conserved G-protiens in the database.
00:36:48.17 So, they are highly conserved even between bacteria and man.
00:36:54.07 Then there are the heterotrimeric G proteins.
00:36:57.18 These are the proteins that are coupled to G protein coupled receptors
00:37:01.04 which sits in the membrane and transmits a signal to these G proteins
00:37:07.07 which are trimeric proteins consisting of G alpha, G beta and G gamma
00:37:11.29 but where the G alpha protein is the actual G protein and which is about 40kDa.
00:37:16.23 I talked about the Ras superfamily. It's a very large superfamily of proteins, all of 20-25kDa.
00:37:23.16 Then you have the Dynamin superfamily of proteins of about 80kDa more or less.
00:37:29.07 It's very important for certain aspects of vesicle formation.
00:37:35.15 Then you have a very small family which is the Signal Recognition Particle
00:37:41.18 which takes the ribosomal nascent complex and brings it to the membrane.
00:37:48.04 And its receptor is in a very small family but it is also very well known and highly conserved family between bacteria and man.
00:37:56.21 Then you have, for example, the septins which are proteins that form filaments
00:38:03.07 by taking the G-domain and making polymers out of it.
00:38:05.24 There is a large family of that in people, at least.
00:38:09.13 And there are many small subfamilies that were not mentioned in detail.
00:38:14.04 All together, you have a couple of hundred proteins, let's say, in mammalian cells.
00:38:23.10 So, if you overlay the structure of all of these, you see this is a huge amount
00:38:28.26 of colored spaghetti that I am showing you where each color indicates a different protein.
00:38:34.27 And you can analyze them and superimpose them very well by overlaying them on the G domains.
00:38:41.08 So the G domain is totally conserved between these very very different proteins.
00:38:44.16 And, for example, you see down there the green stuff here,
00:38:47.00 that would be Elongation Factor Tu. This protein here, hGBP1 would be a dynamin-like protein.
00:38:53.01 And you have up there the SRP, the Signal Recognition Particle and so on.
00:38:57.23 And to just give you one example, mainly that of EF-Tu.
00:39:03.20 So if you look at the topology...so the green stuff here,
00:39:06.25 all the green elements here would be the normal G domain, the alternating beta strands and helices.
00:39:14.01 Here you have the conserved loop elements, the conserved sequence motifs.
00:39:18.08 But then at the C terminus you also have two extra domains; domain 2 and domain 3.
00:39:23.23 And all together this protein is about 45 kDa large.
00:39:28.18 And again, you see now, if I show you the next movie,
00:39:33.18 you see that now the conformation change that, again, is the canonical way I've introduced you to.
00:39:40.16 Down there are the switches.
00:39:41.19 But, now these switches change the structure of these two extra domains in a significant way
00:39:48.00 and this protein binds aminoacyl-tRNA only in the GTP-bound, compact state (which is this state)
00:39:54.02 and not in the open state that you see after the conformational change.
00:39:57.08 So that indicates to you that the trigger for the conformational change is the same
00:40:02.02 but yet it can lead to very drastic conformational change in, also, other domains.
00:40:10.00 So, let me then come to my conclusions which are the following:
00:40:14.21 G proteins are universal switch molecules
00:40:17.06 and I hope I have convinced you that the principle of how they work is universal.
00:40:24.08 Their G domain has a typical alpha-beta structure.
00:40:28.02 I showed you that as an alternating sequence of beta strands and alpha helices.
00:40:35.05 They work by this canonical switch mechanism which we have called the loaded spring mechanism
00:40:40.12 where the two switch elements are bound to the gamma phosphate by main-chain hydrogen bounds
00:40:45.27 are now released when the protein losses the gamma phosphate by the GTPase reaction.
00:40:50.26 They are very specific (most of them at least) for guanine nucleotide and don't bind adenine nucleotide.
00:40:58.28 This is again, different from adenine nucleotide-binding proteins which are usually not so specific.
00:41:05.11 For example, for us the difference is 10 to the 6-fold at least, if not more,
00:41:10.23 for the difference in affinity for GTP versus ATP.
00:41:15.04 They have a very slow intrinsic nucleotide exchange where the dissociation is very slow
00:41:19.14 and is catalyzed by this factor called GEF.
00:41:22.17 They have a very slow intrinsic GTPase reaction
00:41:25.28 which is, again, catalyzed by the protein GAP (GTPase activating protein).
00:41:30.05 So the whole system of the molecular switch is regulated by these GEFs and GAPs
00:41:38.04 which make the reaction faster and can be regulated in the context of a biological system.
00:41:43.03 And I will show you, obviously, in my next seminar, in detail how these GAPs function.
Suppose scientists have discovered a new Ras-like protein, Arr. They found that mutations in Arr and/or ArrGAP were implicated in a form of cancer.
Suggest two possible mechanisms by which a mutation in either Arr or ArrGAP could result in cancer. For each:
- Describe the mutation
- Explain how the mutation would affect the G-protein cycle
00:00:00.14 Hello. My name is Fred Wittinghofer.
00:00:02.18 I'm an emeritus group leader at the Max Planck Institute for Molecular Physiology in Dortmund, Germany.
00:00:11.08 And this is my second seminar
00:00:14.01 and in the first one I introduced you to the molecular switches called GTP binding proteins or G proteins.
00:00:21.15 And in this second part I will more concentrate on a particular aspect of our research which is
00:00:28.20 dealing with the mechanism of GTPase reactions and how they lead to a number of different diseases.
00:00:34.09 And I will obviously focus mostly on Ras-like proteins that I talked about in my first seminar.
00:00:42.23 So, again, just briefly, the mechanistic cycle for these proteins:
00:00:49.25 They come in two flavors these proteins; the GDP-bound and GTP-bound state.
00:00:55.13 They have nucleotide bound very tightly.
00:00:58.03 They need a guanine nucleotide exchange factor to release GDP for GTP
00:01:03.23 because in the cell, there is more GTP than GDP.
00:01:06.11 That's why the protein become loaded with GTP once GDP is off.
00:01:10.06 They have a downstream affect in the GTP bound form; interacting with some partner proteins.
00:01:15.10 And in order for the switch to be switched off again, you have the GTPase reaction
00:01:20.27 whereby the protein splits GTP into GDP and Pi.
00:01:24.29 And this reaction is very slow and is catalyzed by proteins called GTPase Activating Proteins
00:01:31.06 which will obviously be the major thing that we will be talking about.
00:01:36.24 So what we're talking about, really, is a really basic biochemical reaction;
00:01:43.23 namely, the hydrolysis of phosphoanhydrides and it's similar to the hydrolysis of phosphoesters.
00:01:53.08 For example, when you have phosphorylated protiens which are phosphorylated on the threonine, serine or tyrosine,
00:01:57.24 you have a similar nucleophillic attack on the phosphate by water.
00:02:05.04 And obviously, people have been talking about this reaction a lot because it is a very
00:02:12.15 slow reaction for the reason that the phosphates are highly negatively charged
00:02:19.16 and the approach of a nucleophile, for example a water which is partially negatively charged also,
00:02:25.06 is very, very disfavored. And that's why this reaction is normally very slow.
00:02:30.25 So even though the reaction delivers energy,
00:02:33.17 it is very slow because you have to overcome the very high activation energy
00:02:38.16 which depends on what I just told you about--the negative charges.
00:02:42.04 And the higher the activation energy, you might know from your biophysical courses,
00:02:46.13 that the higher the activation energy, the slower the reaction,
00:02:51.00 because the reaction rate is directly proportional to the activation energy.
00:02:54.18 So, nature, then, uses enzymes to lower the transition state energy
00:03:00.27 and thereby make the reaction faster.
00:03:03.15 And there's an interesting article that I bring to the attention of my students all the time
00:03:07.21 from Francis Westheimer who wrote an article many years ago:
00:03:12.01 Why Nature Chooses Phosphates.
00:03:13.25 Because chemists never use phosphate as a leaving group
00:03:17.13 but in biology it is a very frequent leaving group
00:03:21.16 So, because of just this purpose here, because of just what I showed here
00:03:26.17 the phosphoanhydride bond or the phosphoester bond is kinetically stable
00:03:32.06 in other words you can have your ATP or GTP in water and it stays forever or hydrolyzes very slowly.
00:03:39.22 But thermodynamically it's principally unstable.
00:03:43.00 If you use an enzyme, it lowers the activation energy--you can make this reaction very fast.
00:03:47.14 So that's an interesting article that I would recommend you to read.
00:03:50.27 For example, that's why DNA is stable and that's why ATP is such a wonderful source of energy
00:03:57.12 because it, in principle, delivers energy but is stable in aqueous solution.
00:04:03.28 So let's start then with first of all Ras and its GAP-mediated GTPase reaction because
00:04:10.25 that is really the paradigm for many of the things that have been developed afterwards.
00:04:15.24 So this is the molecular Ras. Its a ribbon representation of the structure
00:04:21.29 of the G domain of Ras.
00:04:24.16 You see its heart shaped because...
00:04:27.17 obviously we like it very much and we solved the structure in Hiedelberg.
00:04:32.00 The city of Hiedelberg's advertisement, Ich hab mein Herz in Hiedelberg verloren
00:04:36.04 which means, I lost my heart in Hiedelberg.
00:04:37.27 But the most important reason it's heart shaped is because
00:04:41.13 people call it the beating heart of signal transduction.
00:04:44.06 And so its one of the most important molecules that regulates
00:04:48.14 important signal transduction processes like growth, differentiation and sometimes even apoptosis.
00:04:54.17 And you probably know a little bit about the signal transduction process because
00:04:59.07 every text book has this version, one of the paradigms of signal transduction chain
00:05:04.01 whereby, for example, a growth factor binds to the cell membrane
00:05:08.27 and binds to its growth factor receptor RTK (receptor tyrosine kinase)
00:05:14.22 whereby this becomes phosphorylated. It recruits the exchange factor SoS
00:05:19.04 which then activates Ras to the GDP bound form.
00:05:22.16 And then, now, Ras interacts with the downstream component which is Raf kinase
00:05:26.15 which is the starting kinase for what is called the MAP kinase module.
00:05:30.24 There, one kinase activates the next downstream kinase which is called MAP kinase kinase kinase.
00:05:36.29 MAP kinase kinase kinase activates MAP kinase kinase and that activate MAP kinase
00:05:40.28 which then goes into the nucleus and activates transcription.
00:05:45.03 So this is a very simplified version of what Ras is actually doing.
00:05:48.16 and this is the first one to be discovered (the first signal transduction).
00:05:54.15 But, now it becomes more and more complicated.
00:05:57.26 This is still a very simplified version but it shows you already
00:06:00.27 the major thing about signal transduction via Ras in that many upstream components come and activate Ras.
00:06:09.18 It's either tyrosine kinase receptors or G-protein coupled receptors
00:06:13.09 or T-cell receptors. All of them can activate Ras.
00:06:16.08 And then Ras can activate, downstream, many components, not just Raf kinase
00:06:20.18 but also a molecule called Ral GEF, PI(3) kinase, PLC epsilon and others.
00:06:26.22 And they mediate a number of signal transduction reactions
00:06:32.12 which somehow are integrated somewhere, let's say in nucleus,
00:06:38.00 by a transcription factor and initiate, when the threshold is right, when the number of reaction is right,
00:06:46.15 it initiates a cellular response which can be proliferation or differentiation or whatever.
00:06:52.03 And I will not deal with any aspect of this because I want to concentrate on the switch-off reaction.
00:06:57.28 So the switch-off reaction is again the scheme you have seen, now, many times
00:07:03.25 but what happens in Ras is also an oncogene;
00:07:06.26 an oncogene that has two types of mutations, either the glycine 12 mutated to any other amino acids
00:07:13.10 or glutamine 61 mutated to any other amino acid.
00:07:16.21 And what this does biochemically is, it blocks the GAP-mediated GTPase reaction.
00:07:23.21 So you can imagine what happens, you have blocked the return to the inactive state
00:07:29.13 and that's why you now accumulate Ras in the GTP bound form.
00:07:33.09 You don't need any of the upstream signaling anymore because Ras is already in the GTP bound state.
00:07:39.03 And now it has an effect that is not regulated anymore and that's why it leads to cancer.
00:07:44.14 So these simple mutations...and that's why, obviously, as structural biochemists
00:07:49.23 it is a very interesting project to ask the question:
00:07:52.00 How can it be that a single point mutation has such dramatic consequences?
00:07:58.19 And its also...obviously since Ras is the most frequent oncogene.
00:08:02.28 About 25% of the people that come to the clinic diagnosed with a tumor,
00:08:09.07 they have a Ras mutation, one of the ones I showed you. At least these are the most frequent ones.
00:08:15.00 So obviously every drug company also are working on trying to inhibit the Ras pathway
00:08:20.29 and Ras signaling as a way of treating Ras-mediated cancers.
00:08:25.28 And there are many approaches that one can think of.
00:08:30.17 I just indicated a few here. For example, you could think of Ras...is farnesylated
00:08:36.25 at the C-terminal cysteine and there's an enzyme called farnesyl transferase that mediates that reaction.
00:08:44.18 There are farnesyl transferase inhibitors that are just in the clinic being tested for their efficacy.
00:08:50.00 But you could also think of maybe inhibiting the interaction with downstream effectors.
00:08:54.15 This is, for example, a structure we determined: the complex between Ras and Raf kinase (or part of Raf kinase).
00:09:00.10 Or you can even think of...and that's what we're working on still...
00:09:04.21 Our dream approach is...the basic feature of oncogenic Ras is that is cannot hydrolyze GTP.
00:09:12.09 Can we think about making small molecules that would induce GTP hydrolysis on oncogenic Ras?
00:09:18.21 This is probably a dream project and we're still working on it to make it feasible.
00:09:25.05 But I will show you, in the course of my seminar presentation, that the reason we still think its possible
00:09:32.23 is that the chemistry of it should not be so difficult.
00:09:36.14 And I hope I can convince you and that you will go with me on that point in the end.
00:09:40.26 So, we are talking about, here, a nucleophilic attack of water on the gamma phosphate of GTP
00:09:49.03 mediated by RasGAP and it produces Ras-GDP and Pi. A simple biochemical reaction
00:09:56.09 but if it doesn't work it leads to very drastic consequences, namely cancer.
00:10:00.21 So first of all (And you have seen that picture also a number of times.
00:10:05.24 This is my introductory slide, always)
00:10:08.09 is that the two amino acids that are most frequently mutated
00:10:13.16 (either of them) in oncogenic versions of Ras, they are very close to the active site.
00:10:18.07 So you see the nucleotide here: that is the gamma phosphate
00:10:22.07 And you see this is glutamine 61 and this is glycine 12 are very close to the active site
00:10:28.10 obviously, as you would probably predict or thought of before,
00:10:32.06 is that they are somehow involved in the GTPase reaction.
00:10:35.22 Now, we'll obviously show you what they actually do and why the mutation leads to a block in GTP hydrolysis.
00:10:43.08 And you also can see here from a surface representation of the Ras active site
00:10:49.12 that if this GTP...so it sits...its bound to the surface
00:10:53.26 and you see the gamma phosphate is still approachable from the outside
00:10:57.03 and that will be important in the context of what I will be talking about
00:11:01.12 and you see also, I talked, last time about magnesium being important.
00:11:05.20 If there is no magnesium around you also have no GTP hydrolysis reaction.
00:11:09.24 So, as always in a mammalian genome, there are not just one RasGAP, but rather, 12 or 13.
00:11:19.08 And I indicated here a few of these.
00:11:24.06 And you see they all contain one particular domain of about 330 residues.
00:11:28.09 This is the RasGAP domain which by itself is able to initiate the fast GTP hydrolysis reaction.
00:11:36.17 And you see that all these proteins are composed of different domains.
00:11:43.03 And some are similar, some are vastly different.
00:11:45.20 And they obviously are regulating Ras in different contexts of the cell
00:11:50.04 due to their different domains that they have in addition.
00:11:53.15 And I will be talking about one, the first RasGAP to be discovered by Frank McCormick which is P120GAP.
00:12:02.06 And I will be talking later on about NF1
00:12:05.05 which is another important element for tumor formation because it is a tumor suppressor gene.
00:12:12.04 So first of all, and again to remind you, the intrinsic GTP hydrolysis is very slow.
00:12:17.23 But, if you add the GTPase activating protein, it is fast.
00:12:21.08 So what I have been doing here is I take radioactive GTP (gamma labelled, for example)
00:12:25.27 and then I measure the production of Pi over time.
00:12:29.26 You see, down there, this reaction (this is at room temperature) is almost negligible.
00:12:35.14 There's almost no hydrolysis of normal Ras at room temperature without GAP.
00:12:40.12 And if you now add a particular amount of RasGAP you see that there is a very fast reaction.
00:12:44.23 Much much faster.
00:12:46.04 And under limiting conditions it's actually about 10^5-fold stimulation of that reaction.
00:12:53.10 So that is a way we want to look at it.
00:12:57.05 We want to analyze how GAP mediates this fast GTP hydrolysis reaction.
00:13:03.14 So, initially, obviously, you ask yourself: What could be the mechanism of hydrolysis
00:13:10.11 and what is the step that is catalyzed by GAP?
00:13:14.02 You can think of Ras-GTP being, in principle, a fast GTP hydrolysis enzyme
00:13:20.14 but it needs to come into a GTPase competent state by an isomerization reaction.
00:13:26.15 And that is very slow and is catalyzed by GAP.
00:13:28.29 Or you could think that the actual cleavage reaction, going from GTP to GDP Pi,
00:13:35.02 is very slow and is catalyzed by GAP.
00:13:38.01 Or you could even think that all of that is still very fast
00:13:41.09 but the release of Pi to product is very slow and that is catalyzed by GAP.
00:13:47.08 For example, if you remember having heard about the hydrolysis of ATP on myosin,
00:13:53.26 myosin is, for example, a very fast GTPase but the Pi release is very slow
00:13:59.00 and needs to be catalyzed by actin.
00:14:01.04 So, in other words, what is the actual step that is catalyzed by GAP in this particular case?
00:14:07.10 And I should say that it is the cleavage reaction itself
00:14:11.12 which is the major point of attack by GTPase activating protein
00:14:16.21 and that only in the presence of GAP do the other reactions
00:14:20.18 become at least partially rate limiting, at least the Pi release.
00:14:24.10 And I'll show you that later on, as well.
00:14:27.29 And although I've shown you that before, I will repeat that again.
00:14:32.12 So what we use a lot in our studies where we do biochemistry with fluorescent nucleotide,
00:14:38.20 we use a mant- or mGDP or mGTP analog
00:14:42.29 where you have, on the ribose, a fluorescent reporter group.
00:14:46.24 So this would be either deoxy ribose or ribose and on the ribose you have bound by
00:14:52.17 these ester bonds, the mant group which is very sensitive to the environment that it sits in.
00:14:59.19 And that's why it always give very beautiful structural changes as I will show you in a minute.
00:15:07.10 We used stop flow for measuring fast reactions because, remember, the GTPase reaction,
00:15:13.29 which is slow in the absence of GAP becomes very fast
00:15:17.15 and so in order to analyze it in detail, we need to use stop-flow kinetics.
00:15:21.16 So what you'll, for example, do: you have Ras labeled with
00:15:25.10 mant-GTP (so it's the fluorescent version of GTP)
00:15:28.16 and you have GAP and you shot them together into a fluorescent-detection cuvette
00:15:34.02 and you have the stop flow up here in order to
00:15:37.17 make sure that you only put a certain amount of liquid from these two syringes into your reaction chamber.
00:15:44.07 So when we do that, when we shot these two things together, we see that there is
00:15:50.09 a fluorescent increase if you use Ras-mantGTP and a decrease.
00:15:54.09 The increase, again, is very fast.
00:15:56.25 It means the two proteins make a complex.
00:15:58.27 And then over time, the complex dissociates because after hydrolysis neurofibromine does not bind to Ras anymore.
00:16:07.18 And all of this is over, as you can see, after one second.
00:16:10.09 So, very fast phospho-transfer reaction in the presence of saturating amounts of GAP.
00:16:15.07 And as a control, we use Ras bound to an analog, GppNHp,
00:16:21.25 where you that between the beta and the gamma phosphate you have an NH group
00:16:25.16 which cannot be hydrolyzed anymore and now you have, also, a very fast increase
00:16:30.17 (which is complex formation) but no dissociation because that cannot be hydrolyzed.
00:16:35.05 So that is also the proof that the dissociation is due to GTP hydrolysis.
00:16:40.21 So one may ask, "In this reaction here, what on GAP, which residues on GAP,
00:16:50.01 are involved in mediating this fast GTP hydrolysis reaction, that 10^5 fold stimulation of the reaction?"
00:16:58.17 And obviously, you were thinking of which residues, which amino acids could do the job.
00:17:03.16 Or is it more than one amino acid?
00:17:05.16 And as a good candidate, we were thinking of an arginine
00:17:08.16 because if you look at another G-protein, G-alpha protein (of the heterotrimeric G proteins)
00:17:15.02 it is known that that consists of two domains;
00:17:18.25 so this blue domain is the G-domain and the red stuff here is a helical extra domain.
00:17:24.21 But in that helical domain you have an arginine which sits right, smack in the active site
00:17:29.24 and it has been shown that that arginine is important for the reaction.
00:17:33.29 And that has been shown, in a very old experiment
00:17:37.20 because there is a bacterial pathogen called Vibrio cholera which induces cholera
00:17:45.29 and what is does is it actually modifies this arginine on this G-alpha protein.
00:17:51.16 You see, you have NAD and the cholera toxin transfers ADP-ribose onto
00:17:58.03 an arginine of G-alpha which is shown here. This is actually from the textbook here.
00:18:02.26 So this a very old reaction. It had been analyzed many, many years ago.
00:18:07.10 And it shows, when you do this reaction, when you block
00:18:10.16 the GTPase reaction on G-alpha protein, there is no GTP hydrolysis anymore
00:18:16.10 and the protein is now producing cyclic AMP,
00:18:19.14 it opens up channels (cyclicAMP-gated ion channels) and then you get all these symptoms of cholera.
00:18:26.19 So again the question is: would arginine be a good candidate?
00:18:29.29 So we looked for conserved arginine in all the RasGAPs that I've shown you
00:18:33.24 and indeed, we found several, most of them didn't make any difference.
00:18:38.06 But there was only one arginine that when mutated shows the following pattern in the reaction.
00:18:43.19 So you, again, have in the green version (this is the wild-type version);
00:18:47.23 fast increase due to complex formation, fast decrease due to dissociation after GTP hydrolysis.
00:18:53.16 And with these mutants here, arginine mutated to either lysine or alanine
00:18:58.11 or anything you'd like to mutate it to,
00:19:00.03 there is again an increase in the reaction but then it stays up there, no hydrolysis whatsoever.
00:19:06.13 Or, at least under those circumstances, up to a second, there is no hydrolysis.
00:19:10.16 Which tells you already, obviously, that this arginine must be essential for the reaction.
00:19:18.02 So far so good. The biochemistry was clear
00:19:21.09 but obviously you don't really know what arginine is doing in the context.
00:19:24.05 You think you have an idea; it may be going into the active site
00:19:28.02 but, again, we have to wait for the structure to tell us really what it does.
00:19:33.21 First of all, though, let me introduce you to another important concept for analyzing phospho-transfer reaction.
00:19:40.12 And that is using aluminum fluoride complexes.
00:19:43.02 You may wonder why such an inorganic molecule would be important
00:19:47.14 but it turns out that if you look at the nature of the transition state,
00:19:51.20 so this would be GTP being attacked by water.
00:19:55.18 You have a transition state where the phosphate now makes a triginal, flat thing
00:20:00.26 where the nucleophillic oxygen and the leaving group oxygen on the other side
00:20:08.22 are the axial ligands of this penta-coordinate phosphate
00:20:12.11 and this transition state can either be very tight (which is then called associative)
00:20:18.16 or very loose depending on the distance here between phosphate and the two axial ligands.
00:20:25.26 And then you end up with, again, tetragonal phosphate (this Pi free phosphate).
00:20:32.08 And it turns out that aluminum fluoride
00:20:36.05 (which has distance between aluminum and fluoride very similar to
00:20:39.23 between phosphate and oxygen and they are also highly electron negative)
00:20:44.26 that this is a very good mimic of the transition state of the phospho-transfer reaction.
00:20:51.02 The only problem was that while, for example kinesin or myosin or
00:20:56.28 many other phospho-transfer enzymes use aluminum fluoride as a mimic of the
00:21:02.07 gamma-phosphate in the transition state,
00:21:04.17 Ras or Rho and all of these Ras-like proteins, never showed that.
00:21:08.25 Here you see that experiment.
00:21:11.19 You take Ras-mantGDP (it has a certain fluorescent emission spectrum with a maximum at around 440)
00:21:19.03 and now you add the GAP and with one there's no change whatsoever to the spectrum.
00:21:24.12 So nothing happens.
00:21:25.11 But if you now add aluminum fluoride, you see that now you get a blue shift first of all
00:21:31.00 and then an increase in the absorption.
00:21:37.11 That means there is a trimeric complex between Ras, NF1 and aluminum fluoride
00:21:42.07 where the aluminum fluoride sits in the gamma phosphate binding site.
00:21:47.10 And that was very instrumental...
00:21:48.25 and by the way, if you now do this in oncogenic mutants
00:21:52.02 which we know does not hydrolyze GTP and do the same experiment,
00:21:55.15 you see, in the presence of aluminum fluoride and Ras, there's no fluorescent change.
00:22:00.06 And now you add NF1 and there's no increase in fluorescence
00:22:04.12 because you have a mutation (Q61L) that does not hydrolyze.
00:22:08.13 You can also take a mutation of NF1, for example with the arginine mutation,
00:22:13.22 and again there would be no change whatsoever.
00:22:16.05 So in other words, what these experiments again tell us is that Ras in an incomplete phospho-transfer enzyme.
00:22:22.22 It needs the presence of a GAP in order to look like a phospho-transfer enzyme
00:22:28.05 and if there's any mutation that blocks the GTPase reaction, either on Ras or on NF1,
00:22:33.22 we also get no aluminum fluoride complex.
00:22:37.09 So obviously, all of this is nice in terms of doing biochemistry
00:22:42.00 but what really mediates the GTPase reaction, the fast one,
00:22:47.22 we think can only be verified by looking at the structure of the Ras and RasGAP complex.
00:22:55.29 This is shown here. So that was the paradigm for analyzing this type of reaction.
00:23:02.06 So you see, for example, in red you see Ras and down in green is the RasGAP domain (P120 GAP).
00:23:09.13 And what you see then, here...if you look very carefully
00:23:12.04 you see that there is a residue from GAP going into the active site.
00:23:17.14 You see that when it comes back...you see that right there.
00:23:21.01 There is an arginine residue pointing into the active site of Ras
00:23:26.04 and what is does is shown in the next slide.
00:23:29.06 First of all, in this slide it shows the aluminum fluoride really is the flat triangle
00:23:34.00 that sits between the leaving group and the nucleophillic water.
00:23:38.07 So there is a mimic of the transition state.
00:23:39.28 And you see then, also, what are the residues that mediate fast GTP hydrolysis.
00:23:45.17 So this would be the phosphate,
00:23:48.14 so we think now that if we take away aluminum fluoride and
00:23:52.06 think of what the real transition state would look like
00:23:55.06 you have the nucleophillic water and you have the transition state
00:23:58.29 where they are bound to the gamma phosphate
00:24:00.25 and the glutamine is fixating the water relative to the phosphate
00:24:05.07 by an acceptor and a hydrogen bond donor interaction.
00:24:09.17 And the arginine (what we call arginine finger) is first of all stabilizing
00:24:13.29 the position of that glutamine and it also delivers this positive charge
00:24:18.03 of the amino group to neutralize charges in the gamma phosphate.
00:24:22.28 So these are the two residues that are really crucial for the reaction;
00:24:27.08 glutamine 61 from Ras and the arginine finger from RasGAP.
00:24:32.05 And this already explains, for example, why mutations of glutamine 61 in oncogenic Ras
00:24:37.19 mess up the hydrolysis reaction because you can imagine if you have, for example,
00:24:42.09 a leucine or whatever here, if cannot do this kind of interactions here.
00:24:47.00 So any mutation of glutamine 61 is oncogenic because it is a direct catalytic residue.
00:24:53.16 So the structure also explains why mutations of glycine 12 are oncogenic.
00:24:58.22 And that also can be easily seen here on this slide.
00:25:02.04 You have glycine 12 which when mutated to any residue makes it an oncogene.
00:25:06.25 And then you see aluminum fluoride being this flat triangle.
00:25:11.08 You see glutamine 61 being there and you see the arginine finger down here.
00:25:16.09 And they're all very very close together indicated by these blue dashed lines
00:25:20.04 which are indicating that these are almost VanDerWaals distance.
00:25:24.04 And now if you mutate, for example, glycine to the smallest possible amino acid,
00:25:28.11 it would be alanine, you see that it would immediately mess up all these residues in the active site.
00:25:34.01 So for steric reasons, there can be no other residue
00:25:36.24 in the active site in the transition state except for glycine.
00:25:43.08 So the message from all of this was, obviously, that there's an arginine
00:25:49.00 that we call the arginine finger that pulls the trigger on the GTPase reaction.
00:25:54.00 Without it, it is very slow and with it, it becomes 10^5 fold faster.
00:26:01.01 We used also...so coming back to the question: what is now rate limiting in the whole process?
00:26:07.00 So the main regulating step of intrinsic GTP hydrolysis is the very slow chemical step, the slow hydrolysis.
00:26:14.29 But using a different technique we now find out that
00:26:17.25 there is another step that become partially rate limiting and I will show you that in a minute.
00:26:23.29 So we use time-resolved Fourier transform infrared spectroscopy.
00:26:28.10 And with that we can do almost atomic resolution.
00:26:33.11 We can look at atoms in the active site on a millisecond timescale.
00:26:37.27 So if you see an infrared spectrum of a protein, which has amide I and amide II bands,
00:26:47.20 which is not very structured information because its a mixture of information on
00:26:54.26 alpha helices, beta sheets and so on and not a great deal of detail can be taken from such a picture.
00:27:02.26 But what we do is we observe, for a reaction...let's say a protein goes from A to B
00:27:09.05 we observe the different spectrum which at least on this absorption scale
00:27:16.19 does not show much of a difference but if you look in more detail...
00:27:20.15 so you see the absorption there is 0.0 and the absorption down here is 0.02,
00:27:25.14 so we see very small but very reproducible changes
00:27:29.21 in the course of the reaction when A goes to B.
00:27:33.02 And we see negative peaks that mean A goes away and we see positive peaks if B comes up.
00:27:40.22 So we can observe things that are lost and things that come up
00:27:43.29 in the course of the reaction and we can follow these by FTIR.
00:27:49.25 And so if you do...so first of all we need to trigger the reaction at a particular time point
00:27:56.25 in order to observe second timescale structural changes.
00:28:03.04 And in order to do that we use, again, a different analog
00:28:07.00 and the different analog is caged GTP which was developed by Roger Goody
00:28:10.22 who is a colleague of mine in the Institute.
00:28:14.05 And this caged GTP is blocked on the gamma phosphate by what is called the cage group.
00:28:20.22 So it doesn't allow hydrolysis but now, with a flash of light, you can cleave that cage group off
00:28:26.21 and now you have Ras-GTP which can then hydrolyze to GDP and Pi.
00:28:31.19 If you do that with let's say Ras without GAP,
00:28:39.11 what you see is that you have a...for example, you see absorbance...
00:28:44.22 so any absorbance that you might know from infrared, shows an atomic vibration in the bonds.
00:28:51.09 For example, you see vibrations for the
00:28:53.15 alpha, the beta, the gamma, which decrease toward the end and become zero after 2 hours, 5 minutes.
00:29:00.29 So the difference spectrum (subtract the end spectrum from the starting spectrum)
00:29:06.16 and then you see only the changes that are occurring during the reaction.
00:29:11.23 And that is quite normal. There is a single exponential decay when Ras hydrolyzes GTP.
00:29:18.21 No big deal.
00:29:20.15 But what is interesting is that when you analyze the GAP mediated reaction
00:29:24.26 because now you don't see a single exponential decay, but you suddenly see intermediates appear.
00:29:30.14 You see peaks that come up and go down and the most important one is
00:29:35.04 indicated by this number 1113. This is the frequency for that particular change.
00:29:39.28 So you obviously have to analyze what is each band doing, what does it belongs to.
00:29:47.06 So what we're doing in order...we have developed these techniques
00:29:52.17 or our colleagues at the University of Bochum with whom we collaborate
00:29:56.03 they have developed techniques to find out what is each bandwidth,
00:30:01.01 what is each frequency...absorbance change...what is it due to.
00:30:03.28 For example, you find for GTP that there is absorbance change coming up with
00:30:10.13 a rate constant k2. So k1 is the photoisomerization, k2 is one reaction and k3 is the next one.
00:30:17.18 And if you do that, you get an increase and a decrease with k3.
00:30:21.12 And then there is an intermediate which is coming up at 1113.
00:30:26.12 It comes up with k2 and decays with k3.
00:30:30.01 There is free Pi coming with k3. From all of that we can obviously conclude
00:30:35.18 that there is a Pi intermediate with this absorption frequency here, 1113 cm^-1
00:30:44.02 which appears with the rate constant k2 and decays with a rate constant k3.
00:30:50.03 So in other words, the release of Pi is now becoming visible.
00:30:54.25 So we see hydrolysis when this Pi peak comes up.
00:30:58.21 And we see decay when it goes away.
00:31:01.01 And you see this here, for example, in real-time.
00:31:03.25 So the absorption change with the rate constant k2...you see the scheme here:
00:31:09.05 Ras when its in the on state goes to the Pi state and you see there is a protein bound Pi
00:31:17.13 that comes up and the Pi band goes down in the course of the reaction.
00:31:21.20 And this is repeated a number of times.
00:31:23.07 And you also so that there is an arginine finger that is coming in and out of the reaction chamber.
00:31:30.00 So we can follow not just protein bands,
00:31:32.16 we can follow the phosphate bands at atomic resolution on a milli second timescale.
00:31:40.26 And that tells us now, all together, that is the message from all of this,
00:31:46.01 that while you have a high activation energy for the intrinsic hydrolysis reaction of 92kJ/mol
00:31:54.01 you now seperate the reaction in two partial reactions,
00:31:57.16 which have a lower activation energy and that's why is makes it so much faster.
00:32:01.08 So you have the first activation energy for the cleavage reaction. which leads to Ras-GDP-Pi.
00:32:08.03 And you have the second step where you have release of Pi and now you have the product.
00:32:12.14 So that is, again, a general theme of enzymology;
00:32:15.12 that an enzyme catalyzed reaction lowers the activation energy
00:32:20.00 not just by lowering one reaction but also by subdividing it into partial steps
00:32:24.28 each of which has a different activation energy.
00:32:29.22 And here you see that this activation energy is 59 kcal/mol and 66 which means
00:32:36.04 that this is a bit higher and that's why this is the partially rate limiting step of the overall reaction.
00:32:45.12 So let me now give you...so that was Ras and how it leads to tumor formation
00:32:50.14 and we analyzed in detail how this function even on a biophysical level
00:32:55.02 but now let me come back to why certain GTPase reactions, when the don't work,
00:33:03.00 how they lead to different types of diseases.
00:33:05.20 Neurofibromatosis is one of them.
00:33:07.27 I already showed you that neurofibromine, the gene product of the gene is a Ras GAP.
00:33:15.09 And there is a disease called Type I Neurofibromatosis. Its what people have cafe au lait spots on the skin,
00:33:22.21 sometimes small tumors on the skin which can sometimes be rather large and very disfiguring
00:33:28.16 which are caused by mutation of deletion of neurofibromatosis gene.
00:33:35.21 And for example, when we worked on the mechanism of GTP hydrolysis, by GAP and NF1,
00:33:43.00 a colleague from the Charite Clinic in Berlin came to us and told us that he had
00:33:48.03 a female patient which dies at teh age of 35 and she has three sons indicated up there
00:33:55.20 which also have the disease. And he analyzed the blood of the patient and then the tumor itself.
00:34:04.22 He found out that there is a mutation in the sequence of neurofibrobromine
00:34:11.29 where the arginine is mutated to proline. You see that down there; arginine is mutated to proline.
00:34:17.13 And obviously I wouldn't tell you that, if it wasn't the catalytic arginine.
00:34:21.19 So it turns out they have a mutation in the catalytic arginine
00:34:25.23 and when you now do the GTPase reaction that I've shown you before,
00:34:30.09 you see that you have, with normal NF1 you have the blue curve here,
00:34:37.17 it means an increase in fluorescence and a decrease with hydrolysis
00:34:43.03 and now if you take this mutation, R to P, you have increase which means complex formation
00:34:48.03 but no hydrolysis and there is another patient that we've analyzed in the meantime
00:34:53.16 again arginine to anything else, Q in this particular case,
00:34:58.09 leads to a block of its ability to hydrolyze GTP on Ras.
00:35:02.17 So mutation of the essential arginine leads to the disease neurofibromatosis.
00:35:08.16 Also, I should point out that there are many other mutations in neurofibromine
00:35:13.22 which also cause the disease which phenotypically very different in many different patients.
00:35:20.26 Let me introduce you now to a different system that is
00:35:24.06 interesting both from a biochemical standpoint but also from a standpoint of a different disease
00:35:29.11 that I will come to in due course.
00:35:33.03 So what I will be talking about is a molecule called Rap which, obviously, has a cognate RasGAP.
00:35:40.16 And Rap is close homolog of Ras and the name derives from
00:35:44.05 the fact that it is highly homologous to Ras because Rap stands for Ras Proximate.
00:35:49.07 Even though it was considered to be a close homolog of Ras, it does something completely different.
00:35:55.14 It obviously has the same cycle between GDP and GTP. So it works as a molecular switch.
00:36:02.00 But it does not have to do with proliferation, as Ras, or differentiation, but rather
00:36:06.10 it is involved in integrin activation, or platelet activation or other things.
00:36:11.19 So the biology of Rap is completely different.
00:36:14.17 Why the biochemistry is so interesting is that Rap is the only homolog
00:36:22.12 or the only member of the Ras super family that doesn't have a glutamine in the position
00:36:27.18 where glutamine 61 of Ras is involved in GTP hydrolysis.
00:36:33.01 So, it misses the residue that we thought was absolutely crucial GTP hydrolysis and here, its not there.
00:36:39.18 And, obviously, the question is why that is so and how does RapGAP
00:36:42.25 then work on this system and how does it stimulate the reaction.
00:36:48.29 So first of all, let me introduce you to the RapGAPs.
00:36:52.24 There are indicated here five of them but there are more in the human genome
00:36:57.23 but these five RapGAPs all contain a domain that is highly homologous
00:37:04.02 which is indicated here by the light blue and the dark blue staining
00:37:08.09 where the light blue stuff is somewhat different to the dark blue.
00:37:12.05 And I will explain that when we look at the structure.
00:37:13.21 So again, the domain organization of all these five RapGAPs is somewhat different.
00:37:17.25 That means they're probably doing their job in a different biological context.
00:37:23.04 The reason its also interesting is that the dark blue homology region is
00:37:28.10 also conserved in a protein called Tuberin
00:37:30.17 which stands for a disease that I will be talking about in the end.
00:37:34.03 So that's why it was also interesting to look at that reaction.
00:37:37.06 And the third reason its interesting to look at that reaction is indicated in the next slide.
00:37:42.19 But before doing that, let me show you first of all that we do, again, a stopped-flow fluorescence assay.
00:37:48.23 We've developed a system where we can look at this reaction in a biochemical way
00:37:53.12 and analyze mutations and the speed of reaction and so on.
00:37:58.13 So here again, we have Rap-GTP interacting with RapGAP
00:38:03.20 You get a large, quick fluorescent increase which is due to complex formation
00:38:07.28 and a decrease due to GTP hydrolysis and dissociation of the product.
00:38:12.25 And it is all over, again, after one second.
00:38:16.03 whereas without RapGAP, the whole reaction would take hours.
00:38:19.25 So, in other words, we again have 10^5 stimulation of the reaction.
00:38:23.15 But the reason this is also an interesting system biochemically and mechanistically,
00:38:29.09 is that there are a number of conserved arginines in RapGAPs and obviously
00:38:35.00 we thought that one of them would be involved in providing an arginine finger to the system.
00:38:39.06 In fact, we mutated all of them to alanine
00:38:42.06 and none of them has any dramatic effect on activity as one can see here.
00:38:48.02 So the worst reaction is still close to .5/second.
00:38:52.02 So in other words, there is not a dramatic effect when you mutate an of the arginines
00:38:55.29 which makes it unlikely that there is an arginine finger involved in the reaction.
00:39:00.23 So the two residues, intrinsic glutamine and the arginine finger in trans
00:39:09.04 which is Ras and Rho and others make the important catalysis are not here in this system.
00:39:17.05 That means the whole chemistry must be totally different.
00:39:21.15 So we looked at the FTIR of the system again and
00:39:26.07 it shows basically the same features even thought their structures are somewhat different.
00:39:31.16 The basic features are that there is a Pi intermediate whose decrease is rate limiting
00:39:37.23 for the reaction and although its chemically somewhat different,
00:39:41.27 as the different absorption spectra show, it is, indeed, kinetically a most important intermediate.
00:39:50.11 But what was interesting, and we found in this particular case, in the RapGAP case,
00:39:56.10 and it was also found in other systems in the meantime,
00:39:58.27 is that the GTPase reaction is reversible,
00:40:01.29 which sounds sort of crazy because it's a downhill reaction.
00:40:05.06 If you take GDP and Pi and the whole system you would never ever create GTP.
00:40:09.22 But what happens is that if you analyze the reaction by
00:40:13.05 using, instead of normal water, O18 water, which is indicated by this black dot,
00:40:17.23 you would expect that in the reaction you get hydrolysis and then you get GDP and Pi
00:40:23.27 which has one phosphate labelled as O18 oxygen-rather, one oxygen labeled as O18 oxygen.
00:40:30.12 But instead of getting a Pi with one O18 oxygen, you get Pi with two O18 oxygens,
00:40:36.24 with three O18 oxygens, and with four O18 oxygens as analyzed here by a mass spec.
00:40:42.11 You see where you get all these four products, analyzed by mass spectroscopy.
00:40:49.27 So, how does this happen?
00:40:51.09 So it happens because on the protein you have this long-lived intermediate
00:40:56.00 with GDP and Pi, sitting in the active site before going into product that can also
00:41:03.10 do a back reaction to make GTP with one oxygen now on the gamma phosphate.
00:41:08.15 Now, if it reacts again with O18 water, you get incorporation of a second O18 into the product.
00:41:15.27 And if it happens again, a third one and a fourth one.
00:41:18.16 So although the overall reaction is downhill,
00:41:22.12 on the enzyme, you're getting a backwards reaction.
00:41:24.15 And you can never get it once Pi is released
00:41:28.21 because then the activation energy for the reverse reaction is too high.
00:41:33.18 So we solved the structure of RapGAP also.
00:41:37.06 This is a two domain structure where one domain, which you see now to the left,
00:41:44.07 is the catalytic domain--the dark blue region in the homology diagram that I showed you
00:41:49.01 and the light blue stuff is the dimerization domain which is not important for catalysis
00:41:53.08 but is there to dimerize the protein
00:41:56.18 for whatever reason that we really do not know.
00:42:00.13 So in analyzing the catalytic domain, obviously, you ask yourself:
00:42:03.10 What are the conserved residues and which of those play an important role in catalysis?
00:42:08.04 The purple helix that I've indicated here is the most highly conserved region.
00:42:12.00 So it's likely that residues on this helix are somehow involved in catalysis.
00:42:18.05 And indeed, you can mutate many of these and you see certain effects.
00:42:21.12 But the most dramatic effect happens when you mutate
00:42:25.01 N290, so an asparagine, sitting on this catalytic helix.
00:42:29.08 When you mutate that, you get the following result.
00:42:33.00 So we call it, by the way, the Asn-Thumb to make a difference to the Arginine Finger.
00:42:39.03 So it's an Asn-Thumb and you'll see in a minute why we call it an Asn-Thumb.
00:42:44.26 So, first of all, if you now look at the mutation that I talked about,
00:42:48.27 so if you take the N290A mutation and analyze the reaction by, again,
00:42:55.09 this fluorescent stopped-flow assay, you see that while wild type makes a complex
00:43:00.25 and then decays into product,
00:43:02.26 the mutation here makes a complex, this is even, by the way, tighter than in the wild type case,
00:43:09.15 but there's absolutely no hydrolysis.
00:43:11.07 The reaction goes on and on. It stays up there and never goes down.
00:43:14.21 Whereas if you make a mutation like H287 to alanine,
00:43:20.22 you see that there is no complex formation because the fluorescence stays down here.
00:43:24.04 So that reaction is dead because it cannot bind
00:43:27.08 but the red reaction is deal because we think that this is the important catalytic residue.
00:43:32.12 We think that from looking just at the biochemistry.
00:43:35.15 Obviously, to know what it does, we need again to solve the structure,
00:43:38.26 which we did. This is the Rap-RapGAP complex.
00:43:43.15 And you see here the red and the green stuff is RapGAP and the blue stuff is Rap.
00:43:49.20 And you see GTP. But what you also see if you look at it in detail,
00:43:54.02 is that there is, again, something pointing from the red catalytic domain of RapGAP
00:43:59.06 into the active site and that is an asparagine.
00:44:01.17 Obviously, it's the asparagine that I've talked about.
00:44:04.20 which pokes into the active site of Rap. That's why we call it the Asn-Thumb
00:44:08.28 in relation to the Arginine Finger in the other systems.
00:44:12.27 And if you look in detail at what happens at the active site,
00:44:17.02 and if you compare it to three other structures of Ras protein and their cognate GAPs,
00:44:26.19 taking Ran and RanGAP, Ras and RasGAP, and Rho and RhoGAP,
00:44:31.18 where Rho and RhoGAP are from Rittinger et al. and the other structures are from us,
00:44:35.14 you see that the other three, these three structures here,
00:44:40.12 have a glutamine pointing towards the catalytic water,
00:44:44.29 which attacks the gamma phosphate indicated here,
00:44:47.09 which is the aluminum fluoride,
00:44:50.01 a transition state which mimics the gamma phosphate that is transferred.
00:44:54.08 And the red structure here has, instead of the glutamine 61, has a threonine
00:45:00.19 which is pointing aways from the active site; has nothing to do with catalysis.
00:45:04.01 Instead, what you see is that the purple helix here inserts this asparagine
00:45:09.19 into the active site just exactly at the position where the others have the glutamine.
00:45:14.23 So the differences here: the three other structures have a glutamine, which is in cis
00:45:19.29 and Rap and RapGAP have an asparagine in trans
00:45:23.17 which does the same thing; namely, stabilizing the catalytic water.
00:45:30.04 And if you look at the surface of the protein,
00:45:34.01 this is Rap surface shown as a surface representation,
00:45:39.07 and added to it is just the helix from RapGAP.
00:45:43.09 It sits on the surface and inserts this asparagine into the active site.
00:45:48.13 So the gamma phosphate peaks out of that hole.
00:45:50.08 And all RapGAP really does is it has on the helix this Asn, it puts it into the active site,
00:45:57.17 and that alone seems to be, at least in a chemical sense,
00:46:03.03 responsible for stimulating the GTPase reaction by 10^5 fold
00:46:07.10 because if you mutate that thing, it still binds alright, but there's absolutely no hydrolysis.
00:46:11.22 So that makes us think about, again, the future of designing anti-Ras drugs.
00:46:18.02 If all it takes is to insert such a residue into the active site,
00:46:22.11 from a chemistry point of view it should be doable,
00:46:24.24 but we obviously have to develop molecules that bind in a correct way
00:46:28.12 onto the surface, which is not so easy and we are still thinking that we might be able to do it.
00:46:35.15 This is, again, coming back to that.
00:46:38.00 So as an approach to anti-cancer drug target,
00:46:42.03 let's find molecules that induce hydrolysis of oncogenic Ras.
00:46:46.20 And from what we have observed with RapGAP we think, we have hope that it can be done.
00:46:54.26 And the third reason why working with the Rap-RapGAP system is that
00:47:03.05 it's connected to a disease called Tuberous Sclerosis, a benign tumor.
00:47:06.19 People come in with hamartomas in many organs.
00:47:09.16 But the most obvious feature and what the name comes from is that
00:47:14.07 people have, when they do an NMR of the brain, they have
00:47:16.29 these sclerotic, tuberous sort of things in the brain that you see in the NMR of the brain.
00:47:24.17 The people have mutation in two proteins called Tuberin and Hamartin.
00:47:30.22 And Tuberin, as I showed you before, has high homology
00:47:34.22 to RapGAP in this dark blue region, this means the catalytic region.
00:47:39.07 And if you now look, for example, where patient mutations in Tuberous Sclerosis,
00:47:44.23 in Tuberin, where they are occurring.
00:47:47.00 And this is an alignment of different RapGAP sequences and they align with Tuberin sequence.
00:47:53.27 You see that most highly conserved region is, again,
00:47:57.09 this thing here (the red stuff) where you have the catalytic helix.
00:48:01.08 And one of the mutations in a patient with Tuberous Sclerosis
00:48:04.22 (and many actually have that mutation) is on
00:48:07.06 this asparagine that we know is the important catalytic residue.
00:48:11.08 So this is another version of what we have seen before;
00:48:14.21 an important catalytic residue is mutated in the disease Tuberous Sclerosis.
00:48:21.19 So let me, in the last five minutes, just give you another disease,
00:48:25.22 just to make sure that it's not just the Ras and Rap system,
00:48:28.28 that there are many diseases where the inability to hydrolyze GTP leads to very many different diseases.
00:48:35.07 So this is Retinitis pigmentosa.
00:48:38.28 People have pigments on the retina. That's what the name comes from.
00:48:42.28 And they lose vision, they lose peripheral vision.
00:48:47.00 So this is a normal person seeing that building and this is a patient with the disease
00:48:52.15 that loses more and more, progressively, his peripheral vision.
00:48:56.00 And then loses complete vision after the disease develops fully.
00:49:01.19 And there are a number of genes mutated in Retinitis pigmentosa
00:49:06.12 that are called RP12x and whatever.
00:49:09.28 And there is one form that is called RP2.
00:49:13.03 It's an X-linked disease where people have mutations in...a lot of point mutations in a protein.
00:49:21.16 We determined the structure of the protein, which looks like this.
00:49:24.24 This is a beta helix domain, and this is another domain.
00:49:27.20 And you see many of the mutations that you then analyze.
00:49:31.06 You find out that what you see is that most of the mutations
00:49:37.16 determine or mess up, probably, the structure because they are inside
00:49:41.24 the hydrophobic core of the protein and you would imagine that
00:49:45.04 they don't do anything to the catalysis or the interaction of this protein.
00:49:50.19 But there are a few mutations, indicated here, for example,
00:49:54.01 E138G or R118 to H, K, or C. So these residues point into solution.
00:50:03.27 So you would think that they do something important for interaction of the protein
00:50:08.20 or in something--that they are involved in what these proteins are normally doing.
00:50:14.20 So we solved, again, the structure of the complex between Arl3.
00:50:19.13 So we knew that RP2 interacts with Arl3, another Ras-like protein.
00:50:24.26 It's called Arl because it's an Arf Related Protein.
00:50:28.04 And we solved the structure of that. So you see this stuff here would be the RP2 in purple.
00:50:37.25 And you see in green would be Arl3 in a GTP bound form.
00:50:41.25 And what you see then, if you, again, look very closely,
00:50:44.26 you see that there is a residue from RP2, right here, which points into the active site of the Arl.
00:50:52.10 So one didn't know what RP2 was doing, but when we solved the structure,
00:50:55.17 we could immediately see that it smells like a GAP because
00:50:59.18 it puts an Arginine Finger into the active site of the other protein.
00:51:05.08 And if you look at the active site in detail you see that this would be GDP, again,
00:51:10.28 this is aluminum fluoride, this is glutamine from Arl3,
00:51:15.14 and these are three residues from RP2--Q116, R118, E138.
00:51:21.26 And, obviously, all of these residues are mutated in retinitis pigmentosa.
00:51:27.15 And you can see that the arginine is doing the same thing that you've seen now many times before.
00:51:32.22 And it is stabilized by these other residues and all three residues, when mutated,
00:51:38.10 mess up the GAP activity of RP2.
00:51:41.26 So here, the structure really told us what the protein is doing.
00:51:44.19 There was no idea about the function and the structure told us, exactly, that this is a GAP for Arl3.
00:51:53.17 So let me now come to the conclusions from what I have been telling you about.
00:52:00.12 First of all, maybe, conclusions about Ras itself because it's the most important oncogene.
00:52:06.14 It's an incomplete enzyme, it cannot hydrolyze GTP very fast.
00:52:11.09 But then comes RasGAP which stabilizes switch II and the glutamine 61,
00:52:16.10 which is the important structural catalytic element and it also supplies an arginine finger into the active site.
00:52:23.04 You have Gln1 mutation--any mutation of glutamine 61 is an oncogene
00:52:28.29 because it misses, then, the catalytic residue (the system).
00:52:33.00 You have glycine mutants which are sterically compromised to do GTP hydrolysis.
00:52:38.21 There is no way that the arginine finger
00:52:42.06 can go into its proper position when there is a mutation of glycine 12.
00:52:46.04 I also showed you that there is a strongly bound GDP-Pi intermediate.
00:52:50.02 And that Pi release in the system becomes rate-limiting.
00:52:54.28 Whereas without GAP, the chemical cleavage reaction is the rate-limiting step.
00:53:00.23 The second conclusion--more general to the Ras superfamily.
00:53:05.01 Ras proteins are all incomplete enzymes.
00:53:07.20 They all hydrolyze GTP very, very slowly.
00:53:09.27 They have cognate GAPs.
00:53:12.02 So each of the sub-family proteins and sometimes even proteins within the sub-family,
00:53:16.24 have a specific cognate GAP that are required for fast GTP hydrolysis, for catalysis.
00:53:22.17 Some GAPs supply an arginine finger
00:53:25.02 and I have shown you now many different examples--Ras and Ran and Rho.
00:53:30.15 RabGAPs deliver an agrinine and a Gln.
00:53:34.23 Some GAPs supply an Asn thumb.
00:53:37.00 I showed you the example of RapGAP and Tuberin probably does the same thing.
00:53:41.03 Pi release is very often rate-limiting.
00:53:44.10 And what is even more important, and that is my message for the whole talk,
00:53:47.19 is that the perturbed GTPase reaction is involved in a number of diseases:
00:53:53.24 cancer, neurofibromatosis, tuberous sclerosis,
00:53:58.03 retinitis pigmentosa and many more that I haven't talked about.
00:54:01.07 Thank you for your attention. But I would...
00:54:03.00 Before I stop, let me first thank the people that have done the work.
00:54:07.18 The oldest story that I have told you about is that of Ras and RasGAP,
00:54:11.22 which was done by three post-docs in the lab, Reza Ahmadian, Klaus Scheffzek and Robert Mittal.
00:54:17.23 The RapGAP story was done by the students Oli Daumke, Astrid Kramer,
00:54:23.14 Partha Chakrabarti and Andrea Scrima.
00:54:26.18 And the RP2 story was done by Stefan Veltel and Karin Kuhnel.
00:54:32.04 And a lot of the movies that I have been showing you are doen by Ingrid Vetter
00:54:36.20 who also runs my crystallography lab.
00:54:38.15 And she was very, very helpful in almost all the projects.
00:54:42.13 And we have...on the FTIR we have collaborations with
00:54:46.03 our colleagues at the University of Bochum which is Klaus Gerwert and Carsten Kotting.
00:54:51.03 Thank you for your attention.
Dr. Wittinghofer received his PhD from the German Wool Research Institute and pursued postdoctoral work at the University of North Carolina where he studied protein modification. He then returned to Germany where he joined the Max-Planck Institute in Heidelberg and continued studies of the structure and function of proteins involved in protein biosynthesis such as… Continue Reading