G-Proteins as Molecular Switches

Transcript of Part 1: GTP-Binding Proteins as Molecular Switches

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.
00:41:49.04