It is my pleasure to introduce the next speaker,
Professor Robert Huber from the Max Planck Institute of Biochemistry in Martinsried.
He received the Nobel Prize for Chemistry in 1988 together with Deisenhofer and Michel for
“the determination of the three-dimensional structure of a photosynthetic reaction centre”.
That also marked the first crystallization and structure analysis of a membrane protein.
I still remember the exciting time, not only for the photosynthesis community but also for biochemistry,
biophysics and medical research in general.
Professor Huber will talk about a related topic:
proteolysis and its regulation and molecular basis.
(Applause).
What you see here is the faith of an emeritus who has not yet learned to say no.
I am very pleased to be in Lindau which is a very beautiful place of my home country Bavaria
to meet students and advertise my field of expertise which is protein crystallography
and perhaps convince some of you to enter this field, too.
I was very pleased to listen to Professor Blobel,
the previous speaker who made a splendid advertisement of protein crystallography.
So I would like to continue with that.
It is a relatively young field of research.
The father of it was Max Perutz working in Cambridge, England.
But it has undergone a major transformation in the last 20 years by the technical and methodical developments
which originate actually in different fields, in gene technology, recombinant proteins,
high energy physics, computing and computer graphics.
I will not speak about protein crystallography but let the results and biological implications
of high resolution protein structures speak for themselves, as you have already so nicely seen in Blobel’s talk.
The system I have chosen is proteases, a field in which I have worked since the late ‘60s.
Of course during my career I have followed many other side lines, worked on other systems,
perhaps the one best known is proteins of photosynthesis
which led to the Nobel Prize 1988 together with Hartmut Michel and my former doctoral student Johann Deisenhofer.
Back to the proteases.
A very simple chemical reaction, hydrolysis of a peptide bond, hot acid or hot base does it as you know,
but in nature there are enzymes.
And the surprising fact is that there are many enzymes.
About 3% of all the genes throughout the animal and plant and bacterial world encode proteinases.
Unique is, apart from the amount of proteinases that we have, the fact of their diversity, structurally and mechanistically.
They are usually grouped into, according to the major nucleophile, we find the serine, the threonine, metalloproteinases.
But also within these groups there is diversity.
A classical one is the common principle mechanism of the trypsin-like protease family and the subtilisin-like protein family.
So totally unrelated proteases but sharing the same proteolysis, mechanism of proteolysis.
Now, the reason for the abundance and the diversity of proteases is their involvement in physiological processes.
One of them I show here.
This is the blood coagulation cascade leading from a wound to a thrombus by a series of activation steps
which involve proteolytic, limited proteolytic activity.
Of course this process is highly regulated.
There are inhibitors, there are components which counteract this.
One I will come to is the protein C as you will see later.
Now, the last component, the component which makes the thrombus by cleaving fibrinogen is called factor II or thrombin.
I come back to that as an example several times later.
So again, the diversity that we see in the protease field is due to the fact
that limited proteolysis is a major physiological regulatory mechanism.
This diversity of proteolytic structure and mechanism is correlated with the diversity of regulation of proteolytic activity.
This is the topic of my talk this morning.
In this scheme I have tried to categorize these various kinds, principle base of regulating protease activity.
I go through one by one showing examples.
I begin with the simplest one, namely the inhibition of a protease by a protein
which is sterically complementary to the substrate binding site.
This we have found is the first example in the late ‘60s, early ‘70s.
Here you see trypsin in a complex with the basic pancreatic trypsin inhibitor which we not only studied as a complex
but also the individual components to find out that there is no structural change except slight peripherals,
external site chain movements, no structural change when these two components combine.
So pre-form to structural complementarity.
Later we looked at other protease classes.
This is the serine protease class, trypsin-like proteinases, here we have the papain-like and here we have the metalloprotease.
We find that principle over and over again.
Pre-formed sterical complementarity, no structural change.
There is some variation in the extent to which the inhibitor mimics the substrate, binds like the substrate.
Here we have the serine protease case.
In the cystein protease case we have only part of the substrate binding site occupied by an inhibitor segment.
In the case of the metalloproteases we have very extensive, so called exocyte interactions.
These inhibitors called TIMPs are much larger than the protease they inhibit, they so to speak engulf it almost.
So very extensive exocyte interactions to make a very strong and specific interaction.
The surprise came in the ‘80s when we looked at the inhibitors of serine proteases which are in our blood.
They are the guardians in our blood, guardians against unwanted coagulation for instance and many other processes.
They are very large proteinase, serine proteinase inhibitors and they occur in various forms.
They occur in an active form which has a complementary binding surface,
complementary to the substrate binding site of the protease but they also occur in a latent form, which is an isomer.
Chemically absolutely identical, but this loop structure here is partly inserted into this beta sheet structure here.
And they occur in a cleaved form.
So after having done their job, they are cleaved by the protease
and then they rearrange in a way that this red segment is inserted into this beta sheet and they occur in the complex form.
The protease first interacts on top here and then cleaves the susceptible peptide bond of the inhibitor
and then rearranges to the other side.
It’s a very much more complex scheme but the initial binding occurs to a form which is sterically complementary
as we saw in the small inhibitors before.
This I skip and come to the next way of regulation, namely the fact that we find proteases which are explicitly specific.
One of them is thrombin itself.
And this is achieved by making a rather complicated and extended substrate binding surface.
This is thrombin with a small inhibitor bumped and you see this steep canyon which marks the substrate binding site
and you can imagine that it accepts only a rather specific amino acid sequence in there.
This has been used, discovered and used so to speak during evolution by blood sucking animals.
They live on liquid blood.
If it would coagulate, it would die.
So there is a strong evolutionary pleasure to develop a very efficient coagulation inhibitor system.
They do have actually extremely specific and powerful thrombin inhibitors.
What we looked at was at these various complexes of thrombin in blue
and the various portent thrombin inhibitors here in the leech, here in a blood sucking bug and here in the tick.
These green or brown or purple molecules perfectly fit into the substrate binding site that we saw before,
but using very different scaffolds.
So nature plays around and makes it perfect.
This is another way of generating specificity, another example, namely almost solely electrostatic complementarity
that we find in the proprotein convertases, the name of this specific one, the most important one in mammalian species is furin.
It is an enzyme which is required to activate hormones, cell surface receptors and other proteins
by cleaving at the polybasic site.
Again this physiological process is misused by pathogens.
These pathogens, you find quite prominent names in the list of bacterial or viral pathogens,
need the action of furin to become virulent.
They need proteolytic cleavage at their coat proteins or to activate anthrax toxin for instance.
Again at a polybasic site.
You can imagine, there is a lot of interest from the pharma side in developing furin as a target, as a drug target.
But there is the other side of the coin, namely the enormous physiological relevance of these ….
I come back to that later.
How is specificity achieved?
This is the molecule ….
The substrate binds up here.
The specificity is achieved by generating a highly acidic binding site, not surprising.
So that it is full of glutamates and aspartates to make the binding site for this oligo-lysine arginine inhibitor.
So a specificity mainly through electrostatics.
This was the only publication so far we were able to place in “bild”, simply because of the importance of this.
Now proenzyme cleavage.
Most of the proteases are synthesised as proenzymes and there are two ways to activate them.
In both ways there is limited proteolysis.
Of a part it folds away and this process may involve or may not involve a conformational change.
Here not and here a conformational change and there are examples of both.
So the simplest one we saw in procarboxypeptidase.
Again we can compare the classical carboxypeptidase structure with the procarboxypeptidase structure, find no structural change.
This is the active site, marked by the sink.
So the pro-part folds away after limited proteolysis cleavage and then simply exposes the substrate binding site, very simple.
This is a variation of the same scheme.
This is kumamolysin, is a protease from a very thermophilic bacterium.
This is the enzyme part, this is the pro-part.
It shows a very extended linking region which follows the substrate binding site.
But not exactly.
So it is not cleaved or it is only cleaved after a signal which is probably temperature increase to rearrange slightly
so that the active sites can work on it and then the pro-part folds away and the substrate binding site becomes free.
Again no structural change in the enzyme part when we compare the active enzyme with the proenzyme.
Let me go back.
This is the proenzyme cleavage….
And this is the … of the serine protein… thrombin, chymotrypsin.
So the biochemical basis of this had been studied very early by Hans Neurath and Earl Davie in Seattle.
They found that a zymogen is cleaved at a specific peptide point which has a sequence arginine, isoleucine
and then this short peptide, the pro-part folds away and the enzyme then becomes active.
So this was the biochemical evidence.
Now we looked at the structures.
This is a more modern version of it with the example thrombin.
But what happens here is exactly the same throughout the trypsin-like serine protease family.
So we have the inactive precursor with the closed loop, specific cleavage
and then the insertion of this new N-terminus here in the middle of the molecule and a substantial rearrangement of the surface.
So the triggering event of this surface rearrangement is the formation of an internal sort linkage.
So as I said this is the classical trypsin activation mechanism.
I come back to that in a different context later.
Another way to regulate or to limit proteolytic action is by colocalisation.
This we find for instance in all of the blood clotting factors of the coagulation cascade.
The structures look like this, this is factor 10, one of the early factors in clotting.
There is an enzyme part, its structure belongs to the trypsin family.
There are linker domains and there is a membrane anchor.
In this way the reaction is limited to two dimensions to the vessel surface.
This is where it should happen.
Another way of regulating which we found in two versions, protease activity namely by cofactor binding,
there are systems where the cofactor, a small or larger protein fundamentally changes the specificity,
the activity of an enzyme, against its substrate.
There are again two principle ways to do that, namely the cofactor binding
just offers a new interaction site with the substrate so that it is bound stronger, it’s bound productively.
Or the other way, that the cofactor causes an allosteric change in the enzyme
and only this change conformation is able to bind substrate.
And we find both.
The first one is a beautiful example of the consequences of these membrane anchors of coagulation enzymes.
This is thrombin which is peripherally bound to the membrane via a Gla domain.
And there is protein C which is one of the regulatory components in the coagulation cascade.
Now, protein C when activated shuts down coagulation.
And it is activated by thrombin.
So thrombin in this case has anti-coagulatory properties,
while of course thrombin we usually associate with its coagulatory properties.
But in that way by activating protein C shuts coagulation down.
Now, what protein C does when it is activated, it simply degrades all the upstream coagulation factors.
Protein C and thrombin do not care about each other at all,
except when there is a third component around which is called thrombomodulin for obvious reasons.
This is again a crystallographic study.
Now we know that it is these three domains of the multidomain thrombomodulin
which are responsible for making a productive thrombin, thrombomodulin complex,
productive in the sense that it is able to activate protein C.
So this is the binary complex of thrombin with the three domains of thrombomodulin.
And this is the ternary complex thrombin, thrombomodulin and protein C which is bound in exactly the way
and the orientation that its susceptible peptide bond, which has to be cleaved for activation
comes to lie at the active site of thrombin.
What you see here is that a very large interacting surface between the thrombomodulin and protein C is made.
And only if that is available, so only the binary complex is able to bind protein C.
Without thrombomodulin the affinity of protein C to thrombin is too low to be productively bound.
As we have also the structures of the individual components we can say there is no conformational change.
So the cofactor just increases the interaction sites.
But there is another way.
And I am back now again with the serine proteases and their activation which I told you already about,
the triggering event of the internal sort bridge and the rearrangement.
There are staphylococci which are quite dangerous bacteria when in circulation, actually lethal in circulation.
They misuse this physiological process and they have a virulence factor which is called staphylocoagulase
which interacts with the inactive pro-form of thrombin and activates it to an activity like active thrombin without this,
what we thought obligatory proteolytic step that I showed before.
This is staphylocoagulase and all alpha-helical protein.
It embraces the prothrombin.
It has an N-terminus with exactly the same sequence that we find in active thrombin.
It sticks it exactly into the binding site where inactive thrombin, the N-terminus is located.
So staphylocoagulase is a cofactor, induces conformational change by simulating the physiological activation mechanism
and activates prothrombin in this case.
This is an example where the substrate may act as an activator itself.
This is some recent work which we did on tryptase, again a quite interesting drug target for some lung diseases.
It has trypsin-like subunits but it forms a tetramer, the active sites are located inside, here.
In that way by tetramerization the access to the substrate binding site is restricted.
You find that principle then in the proteasome to which I will come in a minute.
Tryptase physiologically occurs in two forms:
A beta-tryptase which is active.
Here, by the way, are the active site residues, the serine and histidine and aspartate and this is the substrate docking site.
This is where substrate has to align in order to be productively positioned relative to the serine-histidine diet.
The other form is alpha-tryptase which is inactive.
It’s very, very similar.
The sequence identity is more than 90% but there is a conspicuous sequence change, namely …