Well, good morning and thank you Astrid and I appreciate to be here again.
Now it's, perhaps some of you may experience some kind of a culture shock after Sir Harold Kroto’s beautiful presentation,
the grand view on science, society and sustainability.
Now back to the world of molecules, the more so because what I'm discussing today is the destruction of molecules.
So an expression that is negatively associated but you will see that it is absolutely necessary required to maintain life.
So, I'm sorry about that.
Now Astrid announced me as associated with the Max Planck Institute for Biochemistry.
That’s still true but after my retirement I am busy at other places that you see here.
Now I'm talking about protein destruction, negatively associated expression but absolutely required to maintain life.
Now, proteolysis of peptide hydrolysis is a very simple chemical reaction by boiling acid or base,
which we understand in all details, but the nature uses enzymes.
And it uses many of them.
This is surprising for such a simple chemical process.
Now, what you see here is a large number of families of proteinases, all of which use different mechanisms
and have different three-dimensional structures.
So why do we have so many of these proteinases?
Now we do have so many because they are important and involved in regulation in physiological processes.
So because proteolytic activity may be very dangerous, destroying essential protein molecules in cells or outside cells,
proteolytic activity is extremely tightly regulated at all levels.
Transcription, translation, activation, inhibition and degradation.
What I will describe today is different mechanisms of activation and inhibition.
And what you see here on this cartoon is the various modes, mechanisms of regulation
that we found in our studies by structural biology of proteinase structures and their mechanisms.
So the cartoon, which I will go briefly through, is based mainly on structural biology studies and functional studies.
But it is quite clear that proteinases are central to many diseases and malignancies
and therefore there is a strong applied interest in these structures and their mechanisms
as well for medicine and crop protection.
And I will mention a few of these aspects during my talk with specific molecules and describing.
So let me briefly go through these various types, kinds of mechanisms that we found in our structural studies.
There is the simple interaction of the green proteinase with its red proteinaceous inhibitor.
This was actually the first example of protease regulation that we saw in the late sixties, early seventies and the inhibitor molecules involved were BPTI and the Kazal inhibitors were BUSI.
You heard about BUSI as the paradigm for the development of protein NMR.
These are small protein molecules.
There is regulation by substrate specificity.
Some of the proteases are exquisitely specific, leaving one peptide bond in the millions of peptide bonds they meet
during their lifetime, in cells or outside cells.
And specificity is by an intricate binding surface.
So steric complementarity is required, electrostatic hydrogen bonding.
There is pro-enzyme cleavage.
So most of the proteases are synthesized as pro-enzymes,
which are inactive and activation is by cleaving the pro-part, a limited proteolysis step.
And this may involve a no structural change so the pro-part is just physically blocking the substrate binding site
or it may involve a profound structural change, though there are examples for both of these mechanisms.
There is regulation by of colocalization, I mean both protease and substrate are membrane anchored.
So for instance all of the clotting enzymes are of that type to limit the activity
of the clotting enzymes to the vessel surface, makes great sense.
There is cofactor binding, this blue cofactor binds to the enzyme and provides an additional binding surface for the substrate.
And only when this is available the substrate is stably bound and processed.
There is no other structural change in either of these components
but there is also examples where cofactor binding causes a profound structural rearrangement of the enzyme part
and only this form then is enzymatically active.
And there are these two mechanisms on which I will focus.
Oh my God.
Well what should I do?
So it brings in slides from the end of my presentation.
(Side discussion takes place.)
Well there are these mechanisms on which I will focus today, which we see in the large intercellular protease machines.
They are large aggregates which have the active sites sequestered inside these particles and regulation
is by opening or closing an entry port.
And that is the last example, a very recent discovery of protease regulation in the HtrA family of proteinases
where there is activation by a stress signal and the formation of a cage around the substrate.
Now these are the molecules I am discussing today, all of them very big with molecular weights up to one million
and consisting of many sub-units.
This is a proteasome that consists of twenty-eight sub-unit HslVU,
it's twenty-four sub-units then tricorn with six and DegP with six or twelve or twenty-four.
Now I should say after Kurt’s talk that all of these are one or two orders of making it too large for NMR.
So it needs crystallography, there is no doubt about that.
So let me begin with the proteasome, which we first analyzed as the archaeal species.
Why did we choose the archaeal proteasome?
Simply because it has a simple chemistry.
It has the architecture of the yeast, a human protozoan that is a cylindrical molecule.
It's built up of twenty-eight sub-units but the chemistry is simple.
So all these alpha sub-units which form the outer rings of this molecule are identical, so it's alpha seven
and then the inner rings is beta seven and then a twofold repetition called the particle symmetrical with the sevenfold axis.
So that was the starting point some years ago with the structural biology of the proteasome.
And then of course we went on to the eucaryotic proteasome.
We have chosen yeast but the yeast sequences are very similar to the human sequences a lot, all we derive,
interpret from the yeast structure as well it for the human structure as well.
Now, it has the archaeal architecture but here all the alphas are different so there is alpha one to seven
and there is beta one to seven.
So a substantially higher complexity we see in eucaryotes.
I'll come back to that later.
Now what is the role of the proteasome?
It's very essential in cells for many reasons.
One is that it is the executioner of the ubiquitin pathway discovered and described
by Hershko, Ciechanover and Rose and honoured with a Noble Prize.
So the executioner of this ubiquitination conjugation pathway is the proteasome, which recognizes the ubiquitin label,
unfolds the polypeptide that is labelled and degrades it.
Now, it does more.
It's a housecleaner.
So it recognizes denatured proteins and degrades them into essentially octapeptides.
Now if these denatured proteins or their products are not endogenous so of, for instance, viral origin,
then a downstream machinery recognizes that fact and transports these peptides,
which are called now antigenic peptides to the cell surface in complex,
the MHC class one molecules and triggers t-cell immune response.
There is no t-cell immune response without proteasome.
So it's absolutely essential for life in eucaryotes.
Now this is a cartoon that shows the players.
We have the core particles.
This is where we do have atomic structure.
This is what I am mainly discussing but it is associated weakly with a regulatory particle,
which is equally complex and its functions are to recognize the ubiquitin label unfold the label protein,
which then defuses into the core and is degraded.
Now this is a presentation of this big molecule with about 70,000 atoms, so this is all atom presentation.
It's clear that it's completely in-transparent
so we have to reduce the complexity by just drawing the secondary structure elements
or making a surface representation, cut open particle so we look inside and we see
that the active sides here marked with a covalent inhibitor are located inside
and they are associated with the n-terminal threonine.
So this experiment defined the mechanism of the proteasome as an n-terminal threonine protease.
Now I can pick only a few aspects of this machine and I would like to tell you a bit about the proteasome as a drug target.
Now it was surprising for me and for all of us, I think that the proteasome,
which plays such an essential role in all eucaryotic cells is a drug target.
But this was serendipitously found by using this simple boronate, a peptide boronate,
and finding that this compound is a drug against certain forms of multiple incurable, multiple myeloma.
Now we found by crystallography, what it does, the boronic acid adds, oscillates the n-terminal threonine hydroxyl group.
This finding some years ago and then the fact that this compound is on the market generated a lot of intensive screening,
in particular of large natural compound libraries.
And what you see in yellow here are compounds that had been discovered and given to us and we looked at the structures of them.
You see they are chemically quite diverse.
They have quite different head groups.
This is an aldehydic group, which forms a hemiacetal with the n-terminal threonine.
And otherwise it has some peptidic moiety.
These are the (inaudible 16.18) which have a beta-lactone and oscillate the n-terminal threonine.
This is the epoxymicine which has a beta-epoxyketone group and adds to the hydroxyl group
and the amino terminals forming morpholino, absolutely specific of course for an n-terminal threonine.
This is the TMC compound.
A heavily, dirivitized peptide, cross linked side chains, of quite some interest
because it does not form a covalent bond with the n-terminal threonine.
And we looked at that in order to help further development.
Now, this is the original compound.
So we did the crystal structure of that to find out that not all of these difficult to synthesize,
chiral centers or substitutes are actually contacting the protein and are necessary.
So we did some kind of a retro design to simplify the natural compound and came up with this relatively simple compound,
which is easy to synthesize and almost as good as a natural compound and can be developed further.
This is the most recent of these, a series of natural proteasome antagonists
so it was a discovery by our colleague in University of Zurich, Robert Dudler,
who found that a plant pathogen secretes a virulence factor of this structure.
And you see here it's characterized by an unsaturated amide grouping and we found
that it targets the proteasome, the plant proteasome.
It targets it and covalently binds to the n-terminal threonine via a Michael-Addition to the C4 carbon here.
How here you see the electron density of those.
This is the kind of experimental data that we have.
And what happens in the plant cells is what one sees,
an accumulation of poly-ubiquitinated cyclin B1 is a sign that the proteasome no longer works
and this leads to apoptosis and to the death of this plant cells.
Now, the structure that we saw was in surface representation closed.
So the active sites are inside and the entry and exit ports are closed.
And in fact this is a latent form.
That is how we isolate the molecule and the plaques here are formed by the n-terminal segments of the outer ring,
which is made from the alpha sub-units.
So we now set out to constitutively activate it,
so make a constitutively active mutant by simply deleting one of these n-terminal segments.
So one out of seven.
And what happens is what you see in the electron density here.
So you remove one strain and the structure is completely destroyed.
Of course there is metal in here.
There are six more poly-peptide chains but this is disordered and then allows diffusion of the substrate into it
and the activity jumps up by a factor of thirty.
Now, a few words about evolution.
Now I spoke about the archaeal simple proteasome, seven identical alphas, betas and seven fold axis and a twofold repetition.
The complex eucaryotic, all alphas different, all betas are different.
Now most bacteria do not have a proteasome but they have a related machine
that is called HslVU and minus the sequence has clearly showed that there is a relationship between these
and these sub-units and of course we did the crystal structures and compared and verified what can be deduced from the sequence.
Now, the question is about evolution.
And it's quite clear that the simple bacterial HslVU is some kind of a ur-proteasome or is the precursor of that.
Although we find it in modern bacteria but there is a clear relationship of this HslVU sub-unit with a beta sub-unit
and also the archaeal alpha sub-units are related.
They have this topology of a beta sandwich in green, topped with alpha helixes, but a lot of differences in the alpha sub-unit.
Now, these two then combine.
We know a lot about the mechanism of oligamerization to form the archaeal proteasome.
But how can this evolution into the modern eucaryotic proteasome happen, where all the alphas and all the betas are different.
And we know from genetic experiments that none of these alphas or betas can replace another one.
So how can that be?
An evolutionary jump from all the same to all different.
There must have been intermediates which either have been lost during evolution
or we just haven’t looked at enough chain sequences.
Now, one more of these machines and back to the archaea again to thermoplasma acidophilum.
This is the proteasome which makes octapeptides and there is a tricorn protease waiting
which is almost as large as the proteasome which makes di-peptides and there are aminopeptidases waiting which make aminoasis.
Now, how does the tricorn look like.
A very different architecture you'll see.
So a three, two symmetrical particle.
We look along the threefold axis.
The active sights, we now magnify one of the big sub-units, 1200 aminoasis residues.
The active sights are located inside and we know from experiments
that the substrate enters through these blue beta propeller structures, a narrow tunnel
which requires complete unfolding and the products leave through the other side, through the yellow beta propeller structure.
Now, we also looked at the structure of these further downstream aminopeptidases and we do have good evidence
that these huge molecules made transient complexes,
such that there is a processive degradation of the poly-peptide into amino acids, in one go.
There is no release of intermediates.
So the proteasome makes octapeptides.
The octapeptides then diffuse into the nearby tricorn, which makes di- and tri-peptides,
which wait at the exit of the tricorn and makes the amino acids.
So a huge machine with a molecular weight of several millions.
Now let me come to this very new protease machine that has another quite fascinating feature.
This cartoon shows what is happening, schematically.
So there is the inactive protease, which needs activation by its substrate and the activating,
the stress signal is by a part a c-terminal part of OMP, OMP stands for outer membrane protein.
And then when this binds the protease opens, becomes active and at the same time it assembles around the substrate, forms a shell.
Now, let me go through the structural evidence for that.
Well this is the DegP or HtrA family, a big family, seen in all kingdoms of life and they have in common the protease,
which is a trypsin-like protease, which we know very well of course from these early studies
on the digestive enzymes and it has PDC domains.
PDC domains are domains that have been seen in many other protein family contexts.
They are receptors for c-terminal parts of other proteins.
So this is the basic architecture, the basic construction scheme of this large family.
Now let’s look at the coli version, the DegP, where we know most about.
And first I'll speak about the function, the general function of it.
Now, the first in this slide I have borrowed from my good colleague, Michael Ehrmann in Duisburg-Essen
and from Tim Clausen who is in Vienna.
Now, the DegP is the housecleaner for the periplasmic space in the ground negative e-coli
and in particular it looks at the proper fold of the outer membrane proteins.
These are, of course, as all proteins, synthesized in the cytoplasm,
transported through the cytoplasmic membrane and then are incorporated into the outer membrane.
A lot of things can happen there.
And OMPs then not reaching its final target.
So the stress signal, the signal that the OMP is not properly folded is an exposed C-terminus.
Now, here we look at the structure of an OMP.
It's an integral membrane protein, it's a barrel structure and it has a C-terminal segment, this red segment,
which is part of the barrel.
Now, when OMPs are not properly folded this C-terminal segment sticks out and is the signal for activating the DegPs,
such that it is removed from the periplasmic space.
So DegP is a housecleaner, I mentioned already.
It has a protease activity and remarkably enough it has also the opposite activity.
It has a chaperone activity.
So it sometimes helps misfolded OMPs to fold again.
In particular, at low temperature.
This is a quite fascinating balance of activities, destruction and rescue, in the same molecule, under some conditions.
I will say a few words about that.
Now, this was the first structure of DegP, which we analysed and found that it is in a hexameric form.
So it's a dimer of trimers.
We look along the three or four axis here.
We see in green the tripsene domains and we see in red the PDC domains.
And we already saw in these first crystals two different forms of DegP6, a closed form and an open form,
showing immediately the mobility of the PDC domains.
They may fold up and form this closed structure and move away to form the open structure.
The active sites are, as in all these machines, buried inside.
Now, this DegP6 is inactive.
And it is inactive, and its inactivity is easily explained because the substrate binding site is entirely distorted.
Now, what you see here in grey is trypsin.
It's trypsin with bound substrate and this is the strain to which the substrate docks.
You see that this loop is entirely distorted.
In the inactive DegP6 it's folded over the active site, immediately explaining the inactivity.
Now, activation comes by the stress signal and the stress signal in the C-terminal segment of an unfolded OMP.
This is the electron density of an activated HtrA structure so it shows the stress signal peptide bound to the PDC domain.
And then this causes a series of structure changes at the interface between PDC and protease domain
and this finally leads to opening up of the active site, of the protease,
the trypsin part such it can bind substrate and become active.
A few words about some basic functional properties of DegP.
First it is a processive protease that we can easily monitor by looking at the spectrum of products over time.
Just monitoring the outflow of the peptides taken from a limited time digest and we see there is no change.
This is a clear indication there are no intermediates released.
We have to explain that.
The products are mainly 12-mer and 18-mer, can we explain that, we can.
This cartoon shows what happens.
We just look at the trimer in this oligomer that you will see in a minute.
So this is the inactive form.
There's a PDC domain and there is the active site, closed, of the protease domain.
Now, when the activating peptide binds it causes structure change,
propagated to the active site in the neighbouring protease domains and then cleavage starts.
And let’s look here.
So there is binding, there is activation and there is cleavage at the red segment.
Now, a new C-terminus is generated and that new C-terminus then is just the right sequence for binding to another PDC domain.
So the substrate shuttles between PDC and active sites, stays in the compartment and is completely digested.
So that explains processivity and the distances between the active sites are such
that we can explain the generation of 12-mers and of 18-mers.
Now, when one isolates DegP from E. coli,
then you find it is a 24-mer, and you find it is a 24-mer with an additional bend, which turns out to be an OMP.
This is the crystal structure determined by Krojer and Ehrmann and Clausen quite recently.
It's a 24-mer, forming an open shell and inside here sits the substrate.
We don’t see by crystallography this abstract because it's disordered.
It has many ways to orient itself in the symmetrical cage.
But it has a size sufficient for an OMP, which nicely fits into here.
Now, by looking with an electro-microscope at this preparation, which allows us to determine the shape of this molecule.
But as we know the detailed structure of the components we can model them in and in the MVC that in fact has matter inside
and that matter is a substrate, is an OMP.
So, this is the summary.
We do have the inactive hexamer, we have the active 24-mer.
Inactivity is because of the distortion of the substrate binding site so this L2 loop does not allow substrate attachment.
Now, in the active 24-mer this is moved and there is easy access to the substrate binding.
This is the trigger.
The trigger is as I said the C-terminal segment of an OMP, which binds to the PDC domain.
This summarizes again what I have explained to you.
Let me repeat it.
We do have the inactive DegP, the hexameric form, just one monomer is drawn here.
We have the OMP that is folded, but if it is unfolded then it exposes the C-terminus.
This binds to the PDC domain that triggers a confirmation of change in the protease domain and at the same time it forms a shell,
a 24-meric or a 12-meric shell around the substrate.
Now, the pure substrate faces 24 proteases around it.
It's hopeless, one would say.
It is degraded in here but it has a certain, at least at low temperature, it has a certain chance.
The chances that it refolds in this compartment, that is it withdraws the signalling peptide
and incorporates it into its own body.
If that happens then DegP returns to the inactive conformation and this leads to disassembly of the shell
and we have liberation of an intact healthy OMP.
So this is the chaperone activity.
At high temperature it's mainly protease.
Well, Astrid, there are, well you gave me -.
(If you want, you can have another couple of minutes.) Thank you.
This, well it's, on Monday, when we had this get together with the students around the table,
I was frequently asked about the companies which I had helped to found some years ago, and which were mentioned in my CV.
So, obviously, there was a keen interest of the students, understandably.
Not all can go into academic jobs and look for other opportunities.
And I told them, there are other opportunities, and I helped to generate, at least in two cases,
such opportunities and let me just briefly go through what had happened.
Now, the field of structural biology has matured to such an extent that it does provide a sound basis for businesses.
And it is a rather interesting business.
It's a business at the interface of basic research,
when there are new structures to be determined for medical purposes for instance or crop science.
So, the interface of basic research and application.
Now, the first of these foundations in which I helped was Proteros about ten years ago.
It now is a medium sized, and actually flourishing, company that did not need much investment.
It grows by the income it has and what it does, it offers to customer’s information about protein structures
and protein-ligand complexes for money, of course.
This is important for pharma companies for further development of the compounds they have found in a screening experiments.
So, Proteros offers this and it has the proper technologies, namely protein production usually.
They covalent protein structure analysis and protein-ligand information, this they sell and they live on it.
It's interesting work and certainly a kind of alternative career, alternative to academic jobs.
The second company is a more traditional, modern biotech company,
namely a company that develops a new therapeutic principle for autoimmune diseases.
It goes back to my own work many years ago with the first structure of an antibody molecule that you see here rotating.
Now, an antibody molecule binds antigen to the tips of these arms and it exerts its effective function with this stem part.
You see.
So that was the first kind of experiment.
The other was then a recent additional study, namely to determine the structure of the receptor of an antibody to immune cells.
Now this is an essential process.
Immune cells, so antibodies bind to the antigen, opsonize it, so bacterium for instance,
and this opsonized complex is recognized by immune cells via their receptors.
These are called FC-receptors because they bind to the FC-part of the antibody molecule.
And this was the structure still in the academic environment, but then we thought now there is big use of that,
in particular in autoimmune diseases.
So what we thought about this is an immune cell, this is an antigen antibody complex, binding to the receptor stimulating it.
And in autoimmune diseases this should be inhibited.
And we thought a proper antagonist for that would be a soluble FC-receptor and that was the basis of the company.
We did some animal experiments, which looked quite good and this is now in clinical tests by SuppreMol.
So with this I would like to end and thank you for your attention.