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Practice English Speaking&Listening with: Alfred Wittinghofer (MPI) Part 1: GTP-binding Proteins as Molecular Switches

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Hello, my name is Alfred or Fred Wittinghofer and I'm an emeritus group leader

from the Max-Planck Institute for Molecular Physiology in Dortmund, Germany.

What I would like to tell you today is about a class of proteins

that bind GTP (so GTP-binding proteins) that work as molecular switches.

And I will tell you in my first lecture, how they work

and in the second lecture, how they lead to a number of different diseases that we have studied.

So imagine, for example, that you have a quiescent cell that sits there in the G0 phase.

It doesn't grow, doesn't differentiate and it needs a signal from the outside

in order to start proliferation right away.

And the way it works is that a growth factor hits the cell

and induces a series of reactions indicated here by these arrows.

And then this series of reactions comes to the cell nucleus

where DNA is duplicated and the cell decides to proliferate.

And one of the most important elements in the signal transduction chain is Ras

a GTP-binding protein, one of the leading molecules in this class

and this then regulates cell growth as a molecular switch.

And you can imagine, if this regulation does not work,

and cell growth is uncontrolled, then you have cancer.

And Ras is one important element of cancer formation

and I will talk about that in my second seminar.

In another example, for example, you have a quiescent cell

where you have the actin cytoskeleton marked here in the left part of the pictures

where the actin cytoskeleton is very diffuse in blocks and small stripes and so on

and then suddenly you hit these cells with

a particular class of GTP-binding proteins called Rho

and then you see what happens.

In one case you get stress fibers,

in the other case you get a structure at the cell periphery which are called lamelopodia

or here you get structures that are making long extensions which are called philopodia

which make the cell move or proliferate or differentiate and so on and so on.

And these reactions are also controlled by a GTP-binding protein

and they are called Rho, Rac or Cdc42.

So the question then boils down to the thing,

how do you construct a molecular switch that is reversible,

that can be regulated at any level and then does the thing that it's supposed to do?

And so nature has devised a very large class of proteins called GTP-binding proteins

that comes in two flavors; in the GTP bound state it's on and in the GDP bound state it's off.

So the difference between these two states is a single phosphate.

And to show that this is a very important class of proteins,

we can find more that 38,000 GTP-binding proteins or G proteins, as I would like to call them,

in about 1300 genomes by December 2010.

So that tells you these are really important molecules found in all kingdoms of life

and some of these proteins are the most highly conserved proteins in nature at all.

So, let me tell you about how these molecular switches work.

And I will talking mostly about Ras-like proteins because these are the ones that we work with

and they are sort of the prototype for learning how these are regulated.

So you start out with a signal that comes, for example, like a growth factor

or whatever, that hits, somehow these G proteins, (and I will be talking to you about that later)

and that induces a series of steps that lead to the protein becoming loaded with GTP.

And then it has its downstream effect.

And in Ras-like proteins it works the following way:

these nucleotides are usually bound very tightly (picomolar range)

such that GDP never comes off by itself

but needs the action of a nucleotide exchange factor which is called GEF (guanine nucleotide exchange factor)

which allows GDP to be released much faster

and then allows GTP to bind to the protein.

And now it is active.

And now it can do its effect but you obviously, since it's a molecular switch, you want it to be switched off again.

And the way you do that is not the reverse, not the exchange of GTP for GDP,

but rather it is the irreversible step, the GTPase hydrolysis.

So GTP is hydrolyzed to GDP and Pi and there is another protein that

stimulates that reaction because it is intrinsically very slow

and becomes stimulated by a protein called GTPase Activating Protein or GAP.

And I will be talking about that, obviously, in great detail in my second seminar

because that is where you see a lot of diseases being due to inability to hydrolyze GTP.

So the downstream effector is then something that is mediating the biological effect

and the effector is a molecule that recognizes, specifically, only the active

GTP-bound form and not the inactive GDP-bound form.

So, just to make you familiar with the way this thing can work,

since the cycle of GDP to GTP is regulated by Kdiss or Kd (dissociation) for GDP

or is regulated by the GTPase reaction, which is Kcat or Koff,

you can see that the signal can either increase Kdiss or it can decrease Koff.

In both cases you get an increase in the effect, in the biological effect

because the effect is finally determined, really, by the GEF reaction or the GAP reaction.

And you can quantify this and say that the amount of biological effect

that is coming out of this system is

directly proportional to Kon (to the introduction of GTP)

or is inversely proportional to Koff (to the GTPase reaction).

If you make Kdiss faster, you get more GTP bound to protein

or if you make the GAP reaction slower you also have an increase in GTP.

So, let's now come to how these proteins look, how you recognize them and so on.

So, obviously, are there sequence motifs?

Are there structures or biochemistry--are they similar between these proteins?

Yes, indeed. What I will show you is that you can identify

these proteins very easily from sequence motifs, from structure

and also the biochemistry are rather similar between all these different proteins.

And you can present some of the general rules for recognizing

and working with these proteins which I will do in the next 30 minutes.

So there are, obviously, when you look at a new protein you may have sequenced

and then you compare the amino acid sequence,

if you find these 5 sequence elements, indicated here,

called G1, G2, G3, G4 and G5, standing for G binding motifs,

then you immediately know that you're dealing with a GTP binding protein or G protein.

And these elements are, for example, the first one is the so called P loop

is a motif G 4-times x (which means any amino acid there)

then another conserved glycine, a conserved lysine, S or T,

and this is one of the most frequently occurring sequence motifs in the database

because not just G binding proteins but also ATP binding proteins have this sequence motif.

The second one is just the conserved threonine

and a conserved D x x G as the Switch I and Switch II motifs.

I will show you later on what Switch I and Switch II means.

And lastly there are two motifs, N K x D (G4) or s A k

where only, for example in the last motif, the alanine is totally conserved.

These are the motifs that are involved in binding the nucleotide

and also are involved in the specificity of the nucleotide.

And I should also remind you that there are a few proteins

which are also GTP-binding but they have a different fold and a different sequence and so on.

These are the most famous examples: tubulin or the bacterial homolog FtsZ

which you have heard about in other of these iBio seminars for example from Ron Vale.

And there are also a few metabolic enzymes.

So it's a strange coincidence that almost all the metabolic enzymes

that need energy to catalyze the chemical reaction, that they use ATP and not GTP

and only very few examples, indicated here, use GTP.

So that seems to be as if nature decided that in order to transmit energy it uses ATP

and for the regulation of processes it uses GTP,

except for these few examples here.

So obviously as biochemists we...or lets say a structural biochemist,

that I and my lab is involved in, we would like to

understand the system we're working with and the biochemistry and the biology of it

by knowing the structure because this is usually giving you the most deep understanding of your favorite system.

So, to be brief, for those of you that have never worked with protein structures...

and we're using x-ray crystallography to determine the structure

although there are other methods that I will not be talking about

and there will be actually an iBio seminar series coming up later on.

But, I'll give you a brief introduction

to give you a feeling for how you get at your particular structure.

So you can see that you're not afraid of using the method in your favorite system.

So what you start out with is you have to have pure protein

sometimes a lot or, let's say lots of milligrams of protein.

You crystallize them. Hopefully that works.

And once you crystallize them you put them through an x-ray beam.

And sorry if this is still a German slide

because Mr. Rontgen is the one who discovered x-rays

and we still like to call them Rontgen-strahlung which means x-rays.

And so you shine these x-rays through a crystal and then these x-rays are diffracted.

You sample the diffraction pattern and then you use that to calculate your result.

Just to make sure you understand this slide I put the English version of it down there.

X-rays shone into a crystal and then being analyzed on a detector.

So you end up, by a complicated mathematical calculation,

which is standardized by the way so you don't have to really learn all the details about it,

but under that method you end up with an electron density map of your favorite protein

and now you have to do the really fun part which is build an atomic model of it.

And I give you here an example of a particular part of Ras polypeptide. So the chain runs from left to right.

You see that, for example, this would be a 5-membered ring that can only be proline.

You see, for example, down there, a bifurcated amino acid

which can be aspartic or threonine or valine.

The same one down here.

And there's an aromatic residue up there which has also a little tip on it so that must by a tyrosine.

So in other words, you end up

(and you probably did it while you were looking at the screen)

you end up with your atomic model with the tyrosine, then an aspartic acid,

a proline, and a threonine down there

and if you look into the sequence of your particular protein

you know this must be a certain part of the sequence of your protein.

So you end up then, in the end, with a ribbon model of your complete protein.

And you can see here that this one is composed of alpha helices and beta sheets

and that's why I just call it an alpha-beta protein

which is, by the way, typical for any nucleotide binding proteins

which is an alpha-beta fold.

If you go into the database you will see that.

So, if you now look at the sequence motif that I have indicated to you a while ago,

G1 to G5, you can now see...where can I find them?

And they're actually found only in the loops.

So, for example, if you see the first beta strands...

so this is the N-terminus, the first beta strand, you go through G1 into the other helix down here

and then down into the other loops down here

and you see that G1, G2, G3, G4 are all in loops

which is again very typical for a protein structure--that the actual fold is a very stable entity

and the business part of the protein where you see changes,

where things bind and are released,

where things are hydrolyzed or chemical conversions are happening,

they are happening in loops that combine these structural elements.

And if you look at the structure you see, actually, that

all the conserved elements here are really on just the one part of the structure right here.

And the other part, down there, is probably unimportant

in terms of, at least, the interactions with other molecules.

Just to give you a flavor of some of the motifs that we are dealing with here,

this is, for example, the P loop

which is a connection between the beta strand down there, it goes though a loop and ends up in the helix.

And you see that the first conserved glycine (which has the number in Ras by the way)

and going through the loop. Coming to the other conserved glycine on top, there.

The conserved lysine is here and then a serenine or threonine.

So what you actually see is that the phosphate sits right in the middle of this loop

and that's why it is some how neutralized by the charges in the loop

and this the most frequent sequence motif in the database.

Many ATP-binding and GTP-binding proteins just contain this motif.

For example, you heard about kinesin, about myosin in these iBio structure series,

and they also contain the same type of motif.

This is shown here again in a little more detail.

You can see that the beta sheet that sits right in the middle of this P loop

makes the main chain interactions and lysine interaction

so that the negative charge of phosphate is neutralized by binding into the P loop.

It is also called the polyanion hole

by Georg Schulz many many years ago

when he worked adenylate kinase.

So, now we know the structure of Ras and that was the first structure to be solved

many years ago by us.

So in order to look at different structures

let me first introduce you to the Ras super family of GTP binding proteins

where each of the sub-families is, first of all, defined by sequence.

So proteins within the sub-family are more similar to each other than proteins outside the sub-families.

And if you align them by sequence you also align them by function because

each sub-family is involved in some kind of different function.

For example, the Ras sub-family is involved in general signal transduction reactions.

The Rab family is involved in vesicular transport.

The Rho protein sub-family, I introduced you to already, regulates the actin cytoskeleton.

And I showed you Rho, Rac and Cdc42.

And for example, the Ran sub-family is involved in nuclear transport.

It's a nuclear version of Ras. That's why it's called Ran.

And Rho, for example, is called Rho for Ras Homology.

Rab is called Rab for Ras in the brain and so on.

So all the names derive, actually, from the grandfather of the family, which is Ras.

So we have been working with a number of these proteins

and I will show you a few examples of these

and just, for example, compare their structure.

And we'll also talk about function in my second seminar.

So that indicates that a number of structures have been solved

in the meantime by us and many other people.

And the first correct structure of Ras was in 1989

and we now have about 400-500 deposits in the pdb database.

And not just the protein itself, but also complexes with effectors;

complexes with GEFs and complexes with GAPs.

Obviously, these helped us to understand, actually, the complicated regulation of these proteins

much better than only biochemistry would have done.

So if you look now, let's say, at a few of these structures,

on a first view you would say, immediately, "Yeah, they look totally identical."

which is obviously true. If you, for example, compare Ras and Rap, they are rather similar

and also Rho and Cdc42 and also Rab.

So the overall fold is the same but you see small additional elements.

For example, in the Rab-Rho family you see this extra helix here and there.

And in the Arl family you see an extra N-terminal helix.

But other than that, you see that the structure is the same.

So you would ask yourself, "Why would these proteins do different things if they have the same structure?"

And obviously the answer is very simple.

It's not the fold that determines what these proteins are doing in the biological system.

It's what they're interacting with

and the interaction is determined by the surface of the protein where you have different amino acids.

This can be indicated, for example, by just looking at the charges of the surface

where red means negative charge and blue means positive charge.

And now if you compare, for example, the similar Ras and Rab proteins you see, really,

at the surface it's different enough to

make sure that the Ras protein interacts only with its downstream effectors

and Rap interacts only with its downstream effectors.

And the same is true for the Rho protein which is different from Cdc42

but is also different from Ras and Rap and so on and also Arl and so on.

So the message from all of this is that the overall fold is exactly the same for

all of these proteins but they have additional elements and they have a different surface

that makes these proteins do its particular biological reaction that they are involved in.

So, the next thing that I would like to introduce you to is how the switch works.

So. obviously, in our schemes I have also always shown that the GDP-bound form

is different from the GTP-bound form by a symbol.

But now we will obviously look at the structure and biochemistry

and would like to know: How does this structure actually change

when the protein goes form the inactive to the active conformation.

So the basic element of the switch mechanism that we would like to understand...

how does the protein change structure when it goes from the GDP-bound to the GTP-bound state? the following...most of the structure (which is this grey part down here)

does not change at all when the protein goes from one state to the other.

But there are two elements in the structure called switch I, down there, and switch II, down here

which is part of the conserved elements G2 and G3.

And they contain two amino acids; threonine-35 is the number in Ras

and glycine-60, again the number in Ras

and in different proteins the number would be different.

They are bound to the gamma phosphate by these two main-chain hydrogen bonds

which are indicated here as springs that are loaded by binding to the gamma phosphate.

So switch I and switch II are really important elements of the structure change

and they are obviously called switch I and switch II because

they change their structure when they go from GDP-bound to GTP-bound.

These are the important elements for the biological function because

that's where they change structure when going from one form to the other.

And I will show you a number of examples of how this looks in detail.

For example, in the case of Ras, you see that if you overlay

the structure of GDP- and GTP-bound conformations they are almost totally identical

in most of the secondary structure elements, shown as beta sheets and helices here.

And the only changes, really, are happening in the switch regions,

which are down here in this colored area here.

where, for example, you have a tyrosine-32 that sits inside and goes outside

and you have a threonine down here which goes from the outside to the inside.

So you see these two different changes in switch I up there (which is the purple color)

and the cyan color down here (which is switch II) you see, again, structural changes,

a melting of the helix down there.

So there's a localized change

and this would be the part where, obviously, proteins that

are acting downstream of Ras would be recognizing this part of the structure

which is shown here again in a movie.

You'll see the grey part of the structure doesn't change much.

What is happening is down there.

You see these colored loops that are changing

and this is element where proteins that would recognize this Ras in the GTP-bound conformation

would come and recognize this conformation.

We can show the same thing by coloring the surface of the protein

and you see here in color again the two switches when they go from GDP- to GTP-bound conformations.

And this is actually a simulation of the reaction between the two states.

But what you see again, is that the surface of the protein

where the action happens changes when it goes from one state to the other.

And so that's where the business end of the protein is.

This is where almost all the factors that recognize Ras would attack the protein.

Just to give you a more dramatic example:

So the conformation change is canonical. I showed you that in one of the previous pictures.

But there are some wonderful dramatic changes that we can observe

by looking at different Ras-like proteins,

for example, the protein Ran, that I introduced you to briefly before,

a protein that regulates nuclear transport

and has a the C-terminus an extra element, an extra helix, which we call the C-terminal helix

which is bound by its highly negatively charged end to the positively charged protein.

So it sits on the surface in the GDP-bound state.

Now, see what happens in the GTP-bound state.

You see that now you have the canonical triggering of the switch

down at the gamma phosphate side which would be somewhere here.

The two switches no change their structure and by doing that

they kick out the C-terminal end which does something involved in nuclear transport.

An even more dramatic example is shown by the protein Arl or Arf.

So here you see a protein up in the GDP-bound state.

You see there's alpha and beta phosphate bound but the gamma phosphate

binding site is very far away from where it should be if its in the canonical structure.

So what happens now, if you have a tri-phosphate bound to the protein down there,

it moves what is called the inter-switch region which are actually two beta strands.

It moves it by two amino acids towards the N-terminus

and kicks out the N-terminus and thereby induces a large conformational change

which is shown in a movie again, down here.

So you see these two beta strands that move by a very large distance

and kick out the N-terminal end,

which, the N-terminal end is actually interacting now with the plasma membrane

and does something that has to do with vesicular transport.

So the message from all of these things is that there is a canonical change

when the protein goes from GDP-bound to GTP-bound.

The trigger is the same but the affects can sometimes be very dramatic.

And I showed you another dramatic example for that.

And, incidentally, you have heard a lot in different seminar of this series

that motor proteins like myosin or kinesin also do a conformational change

between the ATP- and ADP-bound state.

And it turns out that the mechanism for that structural change is

almost totally identical to that of G protein.

So you see here, for example, in red on the left side, for the motor protein,

the conserved sequence element.

And on the right you see the sequence elements that I have introduced you to before

which from GTP-binding proteins and they are actually very very similar.

And the conformational change is very similar,

except that in the case of motor proteins it is transmitted to do work.

So, for example, in myosin the conformational change of the switch II

makes a lever arm movement that makes the protein walk along the actin filament.

And in the case of kinesin it walks along the tubulin filament.

And so in Ras this conformational change is obviously converted not into work

but is used to make an irreversible change from GTP-bound to GDP-bound state

by the GTPase reaction.

So let me at the end, or towards the end,

show you some of the biochemical properties of the protein,

in particular, of small G proteins which we usually work with.

So all of them have a high affinity to nucleotides.

High affinity means in the pico molar or nano molar range.

Which means that (and this is the second point here)...

that means that the dissociation of nucleotide is very slow.

The half-life of dissociation at 37 degrees for GDP would be on the order of 20 minutes to 30 minutes.

Which would be way too long for a signal transduction process

where this protein is activated within minutes.

And that's why, obviously, you have these nucleotide exchange factors

that I showed you before.

The third thing that I would like to introduce you to is that the affinity is magnesium dependent.

So in the absence of magnesium, for example, the affinity

of nucleotide is 1000-fold less which is a very interesting technical thing for us.

I will show you that in a minute, why we can work with these proteins.

You can reduce the affinity just by removing magnesium.

The fourth things that's here on the slide is

that there is a very high specificity for guanine nucleotide.

For example, I told you the affinity is in the order of pico molar to nano molar.

But the affinity to ATP or adenine nucleotide is in the order of millimolar.

So, a huge difference in affinity of guanine nucleotide versus adenine nucleotide.

And I'll, again, show you that in a minute, how that comes about.

Also, I showed you already that the GTPase is very often very slow,

again, with a half-life at 37 degrees of 20 to 30 minutes.

And again, in a biological process, you want this reaction to be much faster.

So, actually, almost all G proteins are very slow enzymes, very slow phospho-transfer enzymes.

But, they become, obviously, very good transfer enzymes

when in the presence of the GTPase Activating Protein.

And finally, again, which is a very universal observation, the GTPase reaction

just like the ATPase reactions, are always magnesium dependent.

And I'll show you again what that looks like.

So, you need a divalent ion first to make it high affinity

and for the GTPase reaction.

So for example, how does the specificity of guanine nucleotide come about?

Here you see the two elements that I introduced at the very beginning,

G4 and G5 down there which is N or T KxD which is the G4 motif

or the sAk where alanine is probably the only really conserved element.

And what you see is the guanine base making a very strong, bifurcated hydrogen bond

to this aspartic acid down there which obviously adenine couldn't do.

And also the alanine makes a main chain hydrogen bond with the oxygen

which would be the amino group in the case of adenine and again that couldn't happen.

And you see that the lysine which sits underneath the base

and the alanine make additional contacts.

So that's the reason for the specificity.

And also if you mutate any of the residues around the guanine base

binding site you make the binding very much lower affinity.

So here you see the essential magnesium ion.

It is bound between the beta and the gamma phosphate.

These are two of the ligands of magnesium.

You see there are two ligands coming from the protein itself; a threonine and a serine.

This is always conserved in the GTP-bound state for all GTP-binding protein.

And then you have two water molecules that make the coordination field complete.

And a general theme of any ATPase and any GTPase is that you have magnesium as

a bidentate interaction like this one here where the gamma phosphate is to be transferred

but if you have a cleavage at the alpha phosphate here,

magnesium is sitting between alpha and beta.

So, that's a general thing:

that you need a bivalent ion to neutralize the charges of the phosphate.

And I will talking about that, obviously, in my second seminar

where we'll talk in detail about the GTPase reaction.

Another thing that is very important for analyzing and working biochemically with

these proteins is the high affinity is such that if you isolate the protein, let's say

from E. coli (make it recombinately), that you want to make sure that

you know what nucleotide is bound and to what extent.

So, if you run, for example the protein, on an HPLC column,

you see this peak here at a certain elution volume.

If you compare that to a standardized elution diagram

where you take a mixture of GMP, GDP and GTP in a control reaction you see that this is GDP.

And you can also quantify that because this has been equilibrated

with a certain about of nucleotide such that you now know

that your protein has a 1:1 complex of protein and GDP,

which is important because if you want to analyze the protein

in any of the reactions that this protein does

you want to make sure that you know which protein is bound to it already and to what extent.

People make a lot of mistakes by forgetting that the protein already has nucleotide

and that is important if you analyze the following reaction that I will show you.

For example, you want to do an exchange.

You need to to convert the protein into the form that

you would like it to be in in oder to analyze the following reaction.

And the way you do that is you use EDTA to make a very quick exchange of nucleotides.

So, EDTA picks off the magnesium

and without EDTA you see here that the exchange reaction would be very slow.

So you're looking at the reaction of Ras-GDP and you want to introduce a GTP version of that,

for example, tritium labeled GTP or gamma-P32 labeled GTP.

Here, you see that reaction would take a very, very long time in order to get to completion.

But in the presence of EDTA, it takes minutes

and you have a full conversion of the protein into the GTP-bound form, for example,

if you want to, let's say, analyze the GTPase reaction

because if you start with the GDP form,

the rate-limiting step would be exchange, rather than the GTPase reaction.

And we also use, for example, of fluorescent analogs of GDP, which is shown here.

So you have on the ribose or the deoxyribose (depending on which you want to use)

you have a fluorescent reporter group which is called mant or mGDP or GTP

which is a very sensitive probe for the interaction of the protein with

other proteins, with nucleotide itself and so on.

And we have shown that this fluorescent reporter group does not

disturb most of the reactions of the protein that we are working with.

For example, here, this shows you what a wonderful fluorescent reporter group this is.

So, the lower curve here would be the emission curve of the mant-GDP or -GTP

which is a certain amount of emission spectrum you get

and if you add Ras you see that you get a huge increase in the fluorescence emission

which means, probably, that the probe is now in a completely different environment

and that's why you get such a wonderful change

which you can use now to do lots and lots of different reactions.

Just to again give you an example, you first load the protein with a fluorescent analog

(this is Ras bound mGDP) and now you want to analyze the exchange reaction

with one of the guanine nucleotide exchange factors that I showed you in the beginning.

So, in the absence of an exchange factor, at room temperature,

there would be almost no exchange in the time frame that is shown here (600 seconds).

But now, if you add one of the exchange factors

(which is obviously the catalytic domains of one of the exchange factors),

you see you get a very fast release of nucleotide.

The way we do this is we take a large excess of unlabeled GDP

to replace the fluorescent derivative and thereby, you get a decrease in fluorescence

because when the mant-GDP becomes free, fluorescence is about one half of what it was before.

And you use a very high excess of GDP in order to

make the back reaction spectroscopically silent.

In other words, you can now analyze this by a first order reaction analysis

and say what is Kcat of the exchange reaction of Ras + Sos.

In the very end, let me briefly show you some multi-domain G-proteins

because so far I have talked, mostly, about Ras-like proteins which consist mostly, of just the G domain.

And obviously there are a larger number of G proteins that consist of

many more domains and are much, much larger.

So the proteins that we talked about, the Ras-like proteins are in the order of 20 kDa

but the proteins that are indicated here are much, much larger.

For example, the translation factor...

so these are the factors that regulate ribosomal biosynthesis

which are called EF-Tu or EF-1, EF-G and so on.

So all of these use GTP conversion

for driving some part of the ribosomal synthesis and these are between 40 and 80 kDa large.

And these are the most highly conserved G-protiens in the database.

So, they are highly conserved even between bacteria and man.

Then there are the heterotrimeric G proteins.

These are the proteins that are coupled to G protein coupled receptors

which sits in the membrane and transmits a signal to these G proteins

which are trimeric proteins consisting of G alpha, G beta and G gamma

but where the G alpha protein is the actual G protein and which is about 40kDa.

I talked about the Ras superfamily. It's a very large superfamily of proteins, all of 20-25kDa.

Then you have the Dynamin superfamily of proteins of about 80kDa more or less.

It's very important for certain aspects of vesicle formation.

Then you have a very small family which is the Signal Recognition Particle

which takes the ribosomal nascent complex and brings it to the membrane.

And its receptor is in a very small family but it is also very well known and highly conserved family between bacteria and man.

Then you have, for example, the septins which are proteins that form filaments

by taking the G-domain and making polymers out of it.

There is a large family of that in people, at least.

And there are many small subfamilies that were not mentioned in detail.

All together, you have a couple of hundred proteins, let's say, in mammalian cells.

So, if you overlay the structure of all of these, you see this is a huge amount

of colored spaghetti that I am showing you where each color indicates a different protein.

And you can analyze them and superimpose them very well by overlaying them on the G domains.

So the G domain is totally conserved between these very very different proteins.

And, for example, you see down there the green stuff here,

that would be Elongation Factor Tu. This protein here, hGBP1 would be a dynamin-like protein.

And you have up there the SRP, the Signal Recognition Particle and so on.

And to just give you one example, mainly that of EF-Tu.

So if you look at the the green stuff here,

all the green elements here would be the normal G domain, the alternating beta strands and helices.

Here you have the conserved loop elements, the conserved sequence motifs.

But then at the C terminus you also have two extra domains; domain 2 and domain 3.

And all together this protein is about 45 kDa large.

And again, you see now, if I show you the next movie,

you see that now the conformation change that, again, is the canonical way I've introduced you to.

Down there are the switches.

But, now these switches change the structure of these two extra domains in a significant way

and this protein binds aminoacyl-tRNA only in the GTP-bound, compact state (which is this state)

and not in the open state that you see after the conformational change.

So that indicates to you that the trigger for the conformational change is the same

but yet it can lead to very drastic conformational change in, also, other domains.

So, let me then come to my conclusions which are the following:

G proteins are universal switch molecules

and I hope I have convinced you that the principle of how they work is universal.

Their G domain has a typical alpha-beta structure.

I showed you that as an alternating sequence of beta strands and alpha helices.

They work by this canonical switch mechanism which we have called the loaded spring mechanism

where the two switch elements are bound to the gamma phosphate by main-chain hydrogen bounds

are now released when the protein losses the gamma phosphate by the GTPase reaction.

They are very specific (most of them at least) for guanine nucleotide and don't bind adenine nucleotide.

This is again, different from adenine nucleotide-binding proteins which are usually not so specific.

For example, for us the difference is 10 to the 6-fold at least, if not more,

for the difference in affinity for GTP versus ATP.

They have a very slow intrinsic nucleotide exchange where the dissociation is very slow

and is catalyzed by this factor called GEF.

They have a very slow intrinsic GTPase reaction

which is, again, catalyzed by the protein GAP (GTPase activating protein).

So the whole system of the molecular switch is regulated by these GEFs and GAPs

which make the reaction faster and can be regulated in the context of a biological system.

And I will show you, obviously, in my next seminar, in detail how these GAPs function.

The Description of Alfred Wittinghofer (MPI) Part 1: GTP-binding Proteins as Molecular Switches