Well, it’s a pleasure to be here and to talk to you all.
This is a very wonderful meeting and venue for interacting between the laureates and the students.
What I’m going to do is talk about some of our work on the ribosome and I’m also going to try and say a few words
about how we got into studying this particular problem.
Every set of studies has a pathway and we had a pathway.
So when I was a postdoc in Cambridge, Brian Hartley, who is on the next slide, came up to me in the hallway.
In Cambridge people talked a lot and Brian came up to me and he said, well, and this was about 1969,
the year before I was going to take my faculty position.
He said: “Well, what are you going to do next? What is your next problem?”
And I said: “Well, I thought I’d work on the structure of aminoacyl tRNA synthetase complex with tRNA and aminoacid.”
And he went: “There there my boy, that’s a very good idea,
but why don’t you work on something you could actually do? I suggest hexokinase.”
So I thanked him and I ran down to the library to find out what hexokinase was.
And I then found that Dan Koshland had used hexokinase as one of the basis for proposing induced fit.
Because he was trying to address the question:
Why does hexokinase not hydrolise ATP in the absence of glucose?
After all, a hydroxile group of sugar is just a water mimic and he said:
Ah, there must be an induced fit, the only one the right substrate binds to the catalytic groups, arrange themselves.
And if you don’t have, in this case, both substrates you won’t get the real arrangement.
So we saw the structure of hexokinase with and without glucose and this is one of the structures
that we’ve done in Stryer’s textbook and there is the structure without glucose and there is the structure with glucose.
So right, he was right, that is induced fit.
So then I wanted to turn back to the problem that had been interesting me all along.
And that was inspired by two individuals whom I interacted with a lot, Jim Watson and Francis Crick.
Now, Jim was at Harvard when I was a graduate student, actually I played tennis with him,
went to his group meetings and he was working on the ribosome, so he got me interested in that area.
And when I went to Cambridge, Francis of course was always around talking about everything,
including the ribosome and other things.
So that got me interested in thinking about the central dogma of molecular biology,
which is DNA is copied into DNA, then DNA is transcribed into RNA and then finally the RNA is transcribed into protein.
So we started out working on regulatory proteins and polymerases and did this for many years and we are still doing it.
We are still trying to put together this entire machinery that is involved in replication, transcription,
transcription regulation and the synthesis by the ribosome, and now I’m going to focus on how peptide bond formation occurs.
Now, twenty years after my conversation with Brian Hartley, you probably can’t read it, but this is in 1989,
our structure of aminoacyl tRNA synthetased, gluing tRNA synthetased with tRNA and ATP was published, twenty years.
That would have been a very long assistant professor project.
I don’t think I would have made it.
So it is important to pick important problems, but you have to have the timing right, the right thing at the right time.
So the ribosome, in 1964, when I was a graduate student, this was Jim Watson’s drawing,
this is what the ribosome looked, was known about it.
It had two subunits, a large and a small one and tRNA came into the A side with the amino acid,
but they didn’t know what the tRNA structure was, and then the P side had a peptydyle, a group and didn’t know about the tunnel.
And then there was translocation, so that was the general picture, but not much structure really.
And then Jim Lake, in 1976, did the first EM studies of the ribosome
and showed that the large subunit had this sort of a shape, that’s called the crown view,
and the small subunit had a nice shape and they would snuggle up to each other in a friendly fashion,
but it didn’t say much about function really.
Some years later, actually this was published initially in 1997, by Joachim Frank, he introduced cryo-em,
which I think is a fabulous technique for the present and the future, began to show some little invaginations in the Ribosome.
They approximately positioned the tRNAs and the A side, P side and the E side, but not quite right,
because all these anticodons are together, so that was the beginning of understanding the structure.
So in 1995 we figured that it was time to move on and decide on the ribosome,
because we’d been studying everything else in the central dogma, and you need the right person at the right time.
And Nenad Ban joined the lab in 1995 and said he wanted to work on the ribosome
and he was absolutely the right person at the right time.
And two years later, Poul Nissen joined and the two of them really initiated the project and did a fabulous job.
So what was the problem?
Well, my experience tells me that in an audience like this I’m not in 25 words or less going to tell you the details
of what the problem in phasing is, or for that matter even what phasing problem is,
but we followed the work of the … and the first one to solve a structure of a ... using a heavy atom.
You had to find the heavy atom, but then you have to locate it.
It has to have a large enough signal so that you can measure its effect on the crystal and on the ribosome
we need a super big heavy atom, so we use cluster compounds at very, very low resolution, where the signal is much stronger.
And so here’s Joachim Frank’s twenty axiom resolution map of the large subunit.
The first structure that was published was at nine axiom resolution, that we published, and we could see helixes
and we saw the shape was the same, so we knew we were on the right track and then we went to progressively higher resolutions.
And then finally in 2000, we got the atomic structure with the large ribosome RNA in green and white
and the 5S rRNA coloured in purple and white.
And protein scattered around the surface and the preptydyl transferring centre right at the bottom on the left,
totally surrounded by our … and I’m not going to go into this in detail
but we found that only RNA was at the side of peptide bond formation.
So, as Francis Crick had predicted in 1968, the ribosome is indeed a ribozyme,
that is it’s the RNA that’s involved in catalysis, which makes sense.
If you think about the fact that the machine that makes proteins, the first protein couldn’t have been a protein,
it’s the chicken and the egg problem.
So indeed we showed that was right.
And if you split the ribosome in half, now you can see that there’s a tunnel through it
that’s about 100 Å long and about 20 Å wide and right at the surface of RNA that’s split,
showing there’s pretty tightly packed and that there’s protein that protrudes in towards the centre.
And here is a model of the 70th, that we’ve done recently,
and again you can see the RNA has a very complicated twisted shape with the proteins embedded around.
Here’s the large subunit RNA again, protein scattered around on the surface and the bits of protein that go in
are in stabilising the RNA and here we come back to the A side tRNA.
And the message is going in and being decoded here and the peptide bond formation is occurring in the bottom in the large subunit.
So let me now turn to the source of the regular and catalytic power, and again this was done by a graduate student,
Martin Schmeing, who also made a movie that I’m going to show you momentarily.
And so what he did was, what I think is important in using structural studies to understand function,
he captured every state in the process and took a structure of that and then he put it together in a movie.
So here is the process, the A side substrate with an amino acid,
the alpha amino group attacking the carbonile carbon of the peptidal tRNA,
4-amino touching hetero intermediate was an Oxi-N9, and then going to the product.
Now, he couldn’t, in the large subunit, look at the whole tRNA so he actually had just CCA amino acid,
CCAI peptide and so here are the three tRNAs and the long polypeptide tunnel,
and you can see the proteins coming into the interior.
And so we’re going to focus on this area here, and finally he got the situation right at the side of peptide bond formation
with the attacking alpha amino group in a position to attack the carbon EO carbon,
positioned by a three prime hydroxyl of a sugar of A76 and positioned also by this A.
And so then the question is: How does it work, what’s involved in catalysis?
Well, part of it is orienting the substrates, that’s standard.
Orienting the substrates, very important, but there’s more to it than that.
And what’s involved in chemistry, and that was what we were wondering, ...
Scott Strobel and Rachel Green’s lab removed the 2 prime hydroxyl, the P site here,
so this is the 2 prime hydroxyl of the A and the P site tRNA, because that is about the only thing that could be helping here.
And indeed they found that reduced the rate by 10.000 fold.
And earlier, Andrea Barta, using our structure work in her similar studies,
proposed a shuttle mechanism in which the 2 prime hydroxyls, picking up the proton from the alpha amino group
to facilitate a nuclearphyl attack and then that proton going to relieving 3 prime hydroxyl group.
And all the subsequent structural and bio chemical studies are consistent with this shuttle mechanism.
So what’s involved in a ribosome’s catalytic power are substrate orientation,
this proton shuttle and probably transition stabilisation by water molecule.
Well, I could show you all these detailed structures, one at a time,
but rather than doing that I will show you Martin’s movie with music.
(Movie).
Okay, so that’s the RNA proteins in blue, now we’re focusing in onto the peptidyl transferring centre,
we’re going to land on the peptidyl transferring centre, and on one side is going to be the P loop from that ribosome,
it’s combining the P side substrate, and then the A loop is on the other side.
So now we have coming in here, an analogue of the P side substrate, it’s going to make hydrogen bonds,
there’s the structure by the way, and as the commune carbon is protected by this base, Martin discovered,
now we have the attacking A site substrate coming in.
Again making the hydrogen bond with the A loop and now it’s oriented incorrectly to attack,
but when you get a CCA, Martin found, you get a rearrangement and now it’s ready to attack
and there’s a water molecule stabilising the touch of hetero intermediate.
So now we’re about to have the reaction here.
It’s about to attack, okay.
So there’s the touch of hetero intermediate, the Oxi-N9 interacting with the water molecule
and it breaks down to give the product.
And there’s the product CCA and that’s another crystal structure here.
And now the product is going to go off to the exit site, the E site.
There’s always a little bit of Walt Disney in between the structures here, so this is made up.
So the E site is some distance away, now the thing that’s important about the E site is
it has to discriminate against tRNAs with an amino acid, it only binds the A-slated tRNA.
So it goes into a little pocket and there is no room for an amino acid in this region for, so it only binds the A-slated TRNA.
Okay, and then one last trip from the A site to the P site, and it’s ready to start over again.
So that’s what we know about peptide bond formation.
(Applause).
Actually, Martin went from my lab to working in Venki Ramakrishnan’s lab
and he made another movie with music in for EfTu, you ought to listen to that as well.
He’s great. He has a second career possibility, I’m sure.
So now on the last twelve minutes or so I want to talk about antibiotics,
what we know about how antibiotics bind to the ribosome and inhibit it, and the source of antibiotic resistance
and then what is being done to design new antibiotics, not by us but by a company we founded called Rib-X.
I’m sure you’re all aware of MRSA, Mersa, Methicillin-resistant Staphylococcus Aureus,
which in the New York Times, October 17th, in 2007, four years ago almost, 19.000 deaths in US hospitals alone.
I’m told now it’s around 20.000, or 100.000 worldwide.
So it’s a major problem and it’s not the only resistant bacteria, so this is becoming a major problem that needs to be dealt with.
So the question is how to antibiotics bind to the 50S.
It turns out that the ribosome is, about 50% of antibiotics bind to the ribosome, and most of them bind to the large subunit.
So it’s a major target.
And so this work was done by another postdoc, Jeff Hansen initially,
and then there was more work done subsequently that I’ll mention.
So here’s the tRNA binding site, split away in the interior of the ribosome
and there are some antibiotics that are specifically against TB, that I will mention at the end.
There is an E site inhibitor, chloramphenicol and anisomycin.
I’m actually going to skip over this particular one, and then there are macrolides,
I shall concentrate on the bind further down in the tunnel, erythromycin and azithromycin, z-pack,
I expect many of you have taken that.
These are current antibiotics.
And so how do these bind?
So these studies were done some years ago by Jeff Hanson looking at the binding of a whole group of macrolides
and the macrolide rings, all super imposed, they have different substitutions in making different interactions,
almost all with the RNA.
And it binds, here’s where peptide bond formation occurs, it binds further down the tunnel.
So how do they work?
So again, here’s where the antibiotic binds in red, and green are the residues
whose mutation make the ribosome resistant to the antibiotics and there’s where polypeptide chain occurs, synthesis occurs,
so if you look up the ribosome, there’s where peptide bond formation occurs, residue mutations lead to resistance
and there is the antibiotic, it’s blocking the tunnel.
I like to call it molecular constipation, as a way of getting the point across.
And mutation of this residue to a G reduces the binding constant, so it’s a G in Haloarcula Marismortui,
which is a species we use in an AN E.coli, so they decided that in fact they should mutate this residue,
so there is the A or the G, and you can see that that N2 is right under the macrolide ring, so they got to get rid of that.
So this student in post doc decided to mutate that G to an A in Haloarcula Marismortui, make a pseudo U bacteria if you will.
And in fact now erythromycin binds very well, no problem whatsoever.
And you can see where there’s erythromycin, which could bind either situation,
it only changes the position a little bit due to this pesky N2 that’s on the G.
So that means it doesn’t pack as tightly, which is why the binding constant goes down.
Okay, well, now what?
Well, the question that arose for many years is whether antibiotics bind differently to the archaeal ribosomes
that we were studying, Haloarcula Marismortui and the bacterial ones, because archaeal is a sort of a eucaryote in disguise.
They hung out with eucaryotes for a billion years or so.
And so we thought, of course, they were the same, but there was some disagreement.
So if we compare erythromycin as it’s bound to the deinococcus radiodurance in white and Marismortui in yellow,
the rings are orthogonal, so that didn’t look so good.
So these three, a student and two post docs, actually David is here, used thermos thermophilus and redid the experiments,
now asking on the eubacteria what happens and the long and short of the story is that it binds the same.
Here is erythromycin bound to the 70S, it superimposes exactly on our structure
and doesn’t superimpose on the deinococcus radiodurance model.
And the same thing is true for azythromycin, so we conclude that in fact macrolides bind to the mutated 50S
exactly as they bind to the thermo thermophilus ribosomes.
And that’s important if you’re going to use this information,
because the next thing and last thing I’m going to talk about is how do you use this information to make new antibiotics.
And this work I’m going to talk about is done by Rib-X pharmaceuticals and I’m just a reporter here.
And the idea is to take bits and pieces of adjacently bound antibiotics,
so there are many different families of antibiotics that buy nearby in the peptidyle transferring centre.
And so what they do is they take a piece of one and chemically tie it to a piece of another
with a lot of chemical synthesis and computational analysis to see what’s the right thing to do.
So a lot of computational chemistry involved in this as well.
And so here, for example, is one combination, which led to a compound they call Radezolid,
in which these two compounds are combined and Radezolid has completed phase-two clinical trials successfully.
And then another one is to combine macrolide with groups on what’s called the A site, and that’s looking successful.
And then finally, what’s really exciting, is to just use this whole binding area and construct daynovel
using computational methods, as well as structure-based design to make new antibiotics.
And they have a couple of families of compounds that are getting ready for phase-one clinical trials,
that are active against gram-negative bacteria, as well as all the resistance strain.
So everybody is very excited about that as a new way,
and so it looks like there’s many possibilities for designing new antibiotics.
So this is the class called Oxazolidinone and here are the names of the people from Rib-X who worked on it
and so here is, Linezolid is one that you can get from Pfizer, it’s a good compound but against various strains.
So this is the MIC, minimum inhibitory concentration, and if it’s above four it’s not good,
you only have to look at green and red.
Red is bad, and so influenza, MRSA and so on, but Linezolid goes to Radezolid, the Rib-X compound, and it’s much better.
And so again, different enterococci, again, Radezolid is very good, Linezolid is not.
And then the next generation enhanced macrolide, again, the azythromycin, very bad against many things,
stick it together and you get a new macrolide that works very well.
So I think it’s going to work very very well, and then in the last minute or so
I’m going to point out another direction that’s possible to take and that is to attack tuberculosis, a very big problem.
There are, and this is done by a former graduate student and a post doc,
binding some antibiotics to the 70S and here is the 70S, the A site tRNA, small subunit, large subunit,
and this is where this family of compounds, capreomycin, viomycin bind, and they interact with both
the small subunit and the large subunit right near the decoding centre, and let me just skip ahead quickly,
and look at where it’s bound.
It’s interacting with the large subunit RNA, small subunit RNA,
the tRNA and here is the messenger RNA where it’s being decoded and it locks the tRNA on that orientation.
And so what can you do with that?
I mean, this is nice to understand, how it works, but what can you do about it?
Well, it turns out that two other compounds that Venki’s lab have shown bind to the small subunit,
hygromycin and paromomycin bind near to viomycin.
So what would you do if you had that information?
You’d combine it together and make a new compound,
and so I’m hoping that someone will do that hopefully at Rib-X in the near future
and get a better antibiotic that’s useful against TB, because there are some resistant strains of TB,
particularly XRD that’s resistant to all antibiotics that are coming up, that really need to be addressed.
So my main point at the end is that basic research, we only worked on the ribosomes to do basic research,
but it’s having a consequence that will be useful in designing drugs effective against bacteria that are resistant,
and so at the end let me just thank the people who did this work here,
not entirely all on the screen are those in my group who have worked on the ribosome over the years, some of whom I’ve mentioned.
This was a collaboration with Peter Moore’s group, and here are some of the group in his group,
and there is Scott Strobel group made some of the substrate analogues,
and here are some of the people from Rib-X, and then finally I was able to take 14 guests to Stockholm
to enjoy the festivities which were really fantastic I must admit,
and I was able to take some members of the lab who were pivotal and again I’ll mention Peter Moore,
a long-term friend and pillar of the ribosome community.
He was very important in this work and interactions.
Nenad Ban, who started it, Poul Nissen, who then joined in, Hansen, who did the antibiotic work and Martin Schmeing,
who did the substrate work and my administrative assistant Peggy Etherton who keeps me on the straight
and is my memory chip and makes sure I go to the right meetings in the right place and I’ll stop there.
Thank you.
(applause).