Well, it’s a pleasure to be here once again.
And I’m going to talk about our studies again with the ribosome and eventually trying to understand antibiotic resistance.
I just wanted to say a couple of words on how I...
what my pathway was.
In 1968 Brian Hartley who was an enzymologist in Cambridge at the LMB suggested my first project. There’s Brian.
He came up to me in the hall which is what happened in Cambridge.
And he said: “Tell me, what are you going to do when you go on for your next job?” And I said:
“Well, I want to work on the structure of aminoacly-tRNA synthetase complex with tRNA.”
He patted me on the back and he said: “There, there my boy, you ought to do something you can do. I suggest hexokinase.”
(laughter).
So I thanked him and I ran off to the laboratory to find out what hexokinase did.
And I discovered that Dan Koshland had used hexokinase as a model for why one had to have induced fit.
He asked the question: “Why is it that ATP is not hydrolysed in the absence of glucose
because glucose is just a water molecule with some carbon atoms?”
And he said: “It’s because the glucose...”
He proposed, he hypothesised glucose is inducing the conformational change necessary for catalysis.
So we said: “Ok let’s find out.” And we solved the structure of hexokinase without glucose.
And then we did it with glucose. And indeed it goes (click). And Koshland was right.
But then we decided we had to move forward.
And having been at both Harvard and Cambridge and knowing both Watson and Crick,
I decided it was important to study the central dogma put forward by Crick.
That is DNA gets copied into DNA.
So we worked on replication, the replisome for many, many years.
Gets transcribed into RNA by RNA polymerases.
And is regulated at this stage.
We’ve studied that for many years.
And then finally as you heard in the last talk the ribosome reads out the tickertape
and arranges the tRNase on the ribosome for protein synthesis.
And so I’m going to concentrate on that last stage.
Just as an aside, in 1989 about 20 years after my conversation with Brian Harley
we solved the structure of glutamyl tRNA synthetase complex with glutamine tRNA.
It was very... it was the first time and it was very interesting.
And once again Hartley was right.
You have to do the right problem at the right time.
It’s great to have great ideas but you must do it at just the right time.
So we then decided to start on the ribosome.
This is what Jim Watson told me the ribosome was about.
And when I was at Harvard, large sub-unit, small sub-unit, tRNA in the E-site, P-site, peptide.
And then synthesis occurs. There were a few things that weren’t known then.
Didn’t know the structure of tRNA.
Didn’t know there was a tunnel for the exit.
Didn’t know there was an E-site tRNA.
And structural details were missing a bit.
Then Jim Lake did some studies of the ribosome by electron microscopy, did the individual sub-units, found
that they snuggle up together to form a complex. That was pretty good.
Still didn’t tell you much about molecular detail.
And then Joachim Frank actually in 1995 published this cryo-EM.
The first cryo-EM which is now a method taking off I would say.
But at this point it’s still relatively low resolution.
And the positions of the tRNA were not exactly right. But getting there.
So we decided in 1995 that it was time to move on to solve the structure of the ribosome.
And there were 2 pivotally important individuals, Nenad Ban to begin with and then Poul Nissen
who grew the crystals and started the structure and were important in solving the structure of the 50S ribosomal sub-unit.
This is what the low resolution cryo-EM looked like.
You can sort of make out what the structure is.
There is a person holding a torch on his knees.
You know it’s hard to get molecular detail out of this.
But in 1998 we got our first 9 angstrom resolution map.
That began to show some of the details but had the same shape.
So we knew we were on the right path.
And then we moved to higher resolution.
And in 2000 we got a 2.4 angstrom resolution map.
And there’s the structure that we saw in 2000 of the 50S ribosomal sub-unit.
And it was kind of a surprise to us and to everybody to know that the RNA was so tightly packed.
We had no idea that RNA could be quite so tightly packed.
And there’s the peptidyl transferase centre marked by this inhibitor.
Mostly there’s no protein in the neighbourhood.
And there’s a lot of tightly packed RNA.
Proteins scattered around on the surface.
Now if you split the ribosome sub-unit in half like an apple, you can see the peptidyl transferase centre.
And now you can see the tunnel, about 100 angstroms long.
And in green are proteins that are extending into the interior of the ribosome.
So there are bits of the protein that go into the ribosome and stabilise the structure.
But you can see how tightly the RNA, in white, is packed.
So here is the 70S ribosomal sub-unit with the RNA curved around and making complex structure.
There it is, very complex.
To misquote Watson: RNA is not boring like DNA.
It has very complex structure.
And the proteins are scattered around on the surface.
And the tRNA, the decoding centre as you’ve heard is in the small ribosomal sub-unit.
And the peptide bond formation is in the large sub-unit.
So one of the things we wanted to study was the source of the ribosome’s catalytic power and peptide bond formation.
Crick hypothesised in 1968 that the ribosome was a ribozyme realising of course
that how could you have a machine that makes proteins, be made out of protein before there was a protein.
So he suggested it must be RNA.
And indeed we found that turns out to be true.
I had a marvellous graduate student, Martin Schmeing who initiated this work and did most of the work,
I’ll talk briefly about, and made the movie that you’ll see.
And this is the reaction that’s catalysed by the ribosome.
We have an amino acid on the A-site tRNA.
The alpha amino group attacks the carbonyl carbon of the peptidyl group
and the peptidyl tRNA in the P-site to form a tetrahedral intermediate which then breaks down to give the products.
I’m going to show you some results of the structural work but in the 50S sub-unit we only use CCA amino acid or peptide.
More recently of course one can use the whole ribosome and tRNase and we’ve done that as well.
So again splitting the ribosome 50S sub-unit in half, here is the polypeptide exit tunnel.
Inside the box is where peptide bond formation occurs.
And then without going into all of the many, many structures that were solved of the substrate complexes,
this is eventually what Martin Schmeing and co-workers, what he found in the lab was happening at the catalytic site.
The orientation of the P-site substrate here where the carbonyl carbon to be attacked there.
And there’s the alpha amino group from the A-site substrate in a position to attack the carbonyl carbon
interacting with the 2’ hydroxyl here and oriented by A2451.
So what’s the mechanism?
There’s a lot more known now and I won’t go into the details because it’s too much for this short talk.
But the first step in understanding was Andrea Barta in Austria, her lab had proposed a proton shuttle mechanism
in which a proton went from the alpha amino group of the attacking amino group,
was transferred to the leaving 3 prime hydroxyl group.
Now what we’ve subsequently found is it’s a much larger network of hydrogen bonds,
including water and other side chains.
But there is a sort of proton shuttling going on.
So what’s the source of catalytic power?
Actually like most enzymes the primary source of catalytic power is orienting the substrates properly for the chemistry.
Get rid of that pesky entropy that has the molecules wandering around.
The proton shuttle mechanism contributes chemically to it.
And there may be some stabilisation of transition state, which is done by most enzymes.
There are many steps in ribosome protein synthesis.
And there’s an elongation cycle in which you have the peptidyl tRNA in the P-site,
an empty A-site and an EF-Tu protein factor delivers a tRNA.
And then it leaves and the tRNA with the amino acid is oriented.
And then you get peptide bond formation.
This is a low resolution structure of the process.
But we have to know what the process is.
And then this other factor, elongation factor G, EF-G comes in and causes translocation.
And so the question I have is: how does this happen?
Mostly what’s known is it comes in and it just sort of slips along.
Well it must be more complex than that.
So how does it go from here to here?
What’s causing this change?
What is the EF-G doing?
It’s not just doing this on the fly.
And we’ve discovered that’s true.
Now with Peter Moore, a very important collaborator over the years on this project, we solved the structure EF-G by itself.
But more recently we’ve solved the structure of complexes.
Now the Ramakrishnan lab solved this structure of the complex of the EF-G in the post translocation state.
We’ve solved the structure recently.
Hopefully we can find a journal that’s willing to publish it
because it’s so novel and you know you have to be...
They don’t want to publish anything like this.
And the compact confirmation.
So this factor, the components of the factor have moved dramatically from where they are in the elongation complex.
If you look at 4 and then see where 4 moves to.
It’s moved a lot. And so then the question is: What’s happening?
You know these 2 very large changes in the structure.
And what’s happening? Well, look it’s swinging in like that.
And so the thought is that maybe, maybe the conformational change is causing the translocation.
We can make a movie of that with the thing moving.
But we just conclude that it’s the conformational change occurs
in going from that pre-translocation state to the post translocation state that pushes the tRNase along to the next stage.
So my last 10 minutes or so I’m going to turn my attention to again antibiotics and antibiotic resistance.
And you’ve seen many articles about the problems of antibiotic resistance and the resulting deaths due to it.
And the question is how do antibiotics bind in 50S ribosomal sub-unit and what are the mechanisms of the resistance.
You’ve just heard some thoughts on that matter.
Our work was done initially by Jeff Hanson a postdoc and then subsequently by others.
And the tRNA binding site here split through the ribosome.
There are different locations for antibiotics that we’re going to talk about.
The tuberactinomycins here, capreomycin and viomycin bind near the decoding centre.
And they’re important for TB.
They’re A-site inhibitors, which I won’t talk about.
And then the macrolides are a little bit about a bind down the tunnel a bit.
And we’ve looked at many of them but I’ll just talk to you about a couple, just to give an example.
The macrolides, there are different size macrolide rings.
They all bind in the same place down the tunnel.
They have different substituents which interact with the ribosomal RNA in different ways.
There can be changes in the side chain basis from the ribosome.
But mostly it’s pretty steady.
And this is where peptide bond formation occurs.
So what the macrolide is doing is blocking the process.
So how does it work?
Here again looking through the split tunnel, there’s the binding site.
The macrolide is in red.
And in green are bases whose mutation gives rise to resistance.
And you can see that they’re all interacting.
This group is all interacting with the macrolide.
And so you might assume and I think correctly that it’s the change in the shape of the binding site that stops the binding.
Now this is in the absence of the macrolide.
And this is in the presence of the macrolide.
We’re looking up the tunnel.
And so how does the macrolide function?
It’s functioning by what I call molecular constipation.
It blocks the egress of the polypeptide down the tunnel.
So the mutation of A2058 to a G lowers the binding constant by 10,000 fold.
And it’s a G in haloarcula marismortui and it doesn’t bind a lot of antibiotics, that one.
And if we look at where the macrolide binds, you can see that the N2 of that G is right under the macrolide ring.
To look at this Daqi Tu and Gregor Blaha mutated the G to an A in the haloarcula making it a pseudo bacteria.
And the binding constant changed by many orders of magnitude.
We could see where it bound.
And in fact it binds exactly in the same place, the same orientation,
except that the macrolide ring is displaced presumably due to this pesky N2 that comes into the position.
So what do we do with this information?
There are different families of antibiotics that bind to many sites and nearby sites as you’ve heard in the last talk.
So here the binding sites near the peptidyl transferase centre are close together.
And so what’s being done is a company which started out being Rib-X founded by a number of us
who were interested in this area and now Melinta Therapeutics are using these structures to design new antibiotics.
And the slides I’m going to show now are all theirs.
I’m just the conveyer of the information.
So here again is the peptidyl transferase centre with different families of antibiotics
all bound near to each other in the peptidyl transferase centre.
And so what you want... The strategy is, is to take a piece of one and chemically tie it to a piece of another one.
So here for example are 3 antibiotics.
And the first one called Radezolid has quite increased potency.
Actually I’ll mention a bit more about that.
It’s again combining fragments here.
And then you can also combine the macrolide.
And most importantly -and I can’t...
I have no slides to show what they’ve actually done- is to take this whole area and design a whole new family of antibiotics.
And that’s looking very promising work against gram positive and gram negative bacteria.
So this RX-01 family. This takes Linezolid which has no potency against a number of strains.
Red is bad, anything over 4 is bad, very bad.
If you make the Radezolid, the combo, it works very well.
And also is true looking at various enterococci.
You see that Linezolid is not so great.
RX-02, that’s the enhanced macrolide.
The same thing.
If you look at the macrolide, the azithromycin very bad, very, very, very bad, 128 is nothing against all these strains.
Make the combo and it’s very good. So the strategy works.
And I should just say that it works and it’s being developed.
The Linezolid has completed phase 2 clinical trials.
And other things are along the way.
And then just one last thing about the A-site binding sites and this is our work
showing that the viomycin and capreomycin bind to the A-site.
And it binds to the small sub-unit.
And there is the binding site.
It’s interacting with large and small sub-units.
And this shows where it’s located.
It interacts with the tRNA of the large sub-unit and the small sub-unit.
And here is where the decoding happens.
So it’s locking the ribosome in this position.
So it can’t move forward.
Ok, that’s nice to know.
But what are you going to do with it?
Well, it turns out there are many compounds now that are known that bind in this neighbourhood.
There’s the viomycin here but there are many other compounds.
And so if one could make money off of a TB antibiotic, not so obvious,
and if one could get a drug company to develop it, I speculate that one could put these together in some way or other.
Or to just use the surface to design a new compound that would work against TB.
So the idea is that you could use this information to create an antibiotic
that would be active against all these resistant strains.
And this is a major problem because the XDR strain is resistant to everything.
So this is a major point that, a major closing point that I want to make.
And that is that basic research is very important.
Basic research on the structure and function of the ribosome
is leading to the development of new antibiotics that are proving effective against resistant bacterial strains.
And there’s a lot of push in the US and other places for the NIA to support translational research.
And I have to say the only translational research I’m in favour of is the ribosome.
And so I’ll stop there.
Thank you.
Applause.