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ELIZABETH NOLAN: --by talking about ClpX.
And then we're going to move into module 4--
which is the last module before spring break--
on synthases and assembly line biosynthesis.
So basically last time, where we left off is,
we went over experiments that were
done to look at denaturation, translocation,
and degradation by ClpXP.
And closed with a question about what actually is going on
in ClpX with this ATP binding and hydrolysis
to allow for these condemned protein
substrates to be unfolded and translocated
into the degradation chamber.
And I left you just with the statement that although we
think about ClpX as this hexamer that has six identical
subunits, what studies have shown is that there's some
inherent asymmetry within this AAA+ ATPase.
And that's what we're going to talk about a little bit.
So this is just a slide from a few lectures ago
that's showing the top view and side view of ClpX
and how we've thought about this.
And I think these studies just really
highlight how complicated these machines are
and that there's still a lot more we
need to figure out here.
So as I said last time, this asymmetry
comes from whether or not each ClpX subunit
is bound to nucleotide.
And so basically, from looking at many different crystal
structures, what can be done is that the ClpX subunits
can be divided into two different types based
on conformation here.
And so in thinking about this, we
want to first think about the ClpX domain organization.
And if we just think about this, what ClpX has is
an end domain followed by a domain
that's called the large domain and then followed
by a small domain.
So 633 amino acids, just to give you a sense of size,
and about 69 kilodaltons per subunit.
And so what we're going to focus on
are the large and the small subunits
and what's observed from many different crystal structures.
And so these two different types of subunit
have been described as loadable and unloadable,
and that depends on whether or not nucleotide is bound.
So if we consider of these two types,
just thinking about the large and small domains,
we have this loadable arrangement
which binds nucleotide.
And in cartoon, something like this.
So we have the large subunit.
We have the small subunit.
And we have this region that's called a hinge.
So this is one ClpX.
So ATP binds.
And so the other type is described as unloadable.
And this type of subunit does not bind
nucleotide when in this unloadable conformation.
And so we can draw this.
Here, again, we have the large subunit.
And there's a change in conformation.
And here's the small subunit here.
So what's been found from looking at many crystal
structures is that within the ClpX hexamer,
there's an arrangement of these loadable and unloadable
subunits.
So in many crystals, what's found
is that there's four loadable--
I'm just going to do with "L"--
plus two unloadable subunits arranged with about
two-fold symmetry, so LULLUL.
So there's some asymmetry in the subunits.
And so also from these crystal structures,
there's some more observations that we
don't see with just these cartoons of the 6-mer.
So we can learn about how subunits interact, of course,
and this is what's shown.
So if we look at these structures
and consider how these subunits interact, what we find
is that the small subunit of one ClpX--
or sorry, the small domain of one ClpX subunit interacts
with the large domain of the adjacent ClpX subunit.
And so we can draw this.
Basically, if we consider a large domain--
and let's say this is subunit 2--
then what we find is that there's
the small domain and then the large domain
of subunit next door.
So let's call this subunit 1.
So here's our hinge of subunit 1,
and ATP binding happens in here.
So we can think about this arrangement.
And then what's been defined is something called a rigid body.
And so this rigid body is comprised
of the large domain of one subunit
and the small domain of the next.
Rigid body.
So large domain of one ClpX and small domain of another subunit
that's adjacent.
So in thinking about this, we can consider the ClpX hexamer
in another way.
So how I've initially presented it to you
when we introduced these oligomers is just as a 6-mer,
right?
6 subunits.
But another way to think about ClpX
is that it's actually six rigid bodies that
are connected by hinges, where each rigid body has
a component from two subunits, a large domain from one
and a small from another.
And so the hinges are within a single subunit
based on this cartoon where ATP binds.
And so the thinking is that ATP binding and hydrolysis
results in changes in the hinge geometry
and that this change in confirmation
in the hinge with ATP binding and hydrolysis
allows for conformational change in another subunit here.
So six rigid bodies connected by six
hinges, effectively, as opposed to just six
standalone subunits.
Each subunit's communicating with one another here.
So this is pretty complicated, right?
It's another level of sophistication
within this hexamer here.
So what about these loadable and unloadable conformations?
I've told you that in these crystal
structures, what's seen often are these four
loadable and two unloadable subunits
with a particular arrangement.
So we can ask the question, do these individual subunits
maintain the same conformation during these attempts
to denature and translocate polypeptides?
So is one subunit just committed to being
loadable and another subunit committed to being unloadable?
Or did they switch dynamically?
And so recently, there were a number
of studies looking at that.
And effectively, as of a few years ago,
many studies suggest--
or support switching by a given subunit.
And they also indicate that every ClpX subunit
must bind to ATP at some point during these cycles
for unfolding and translocation.
But they're not all doing it at the same time.
So the way to think about this is
that there's some dynamic interconversion
between these loadable and unloadable subunits
within the hexamer, and somehow--
yep?
AUDIENCE: I just want to ask you--
ELIZABETH NOLAN: Going to make trouble?
JOANNE STUBBE: --a question.
Yeah.
So when you have all these structures,
are they all with an ATP analogue?
ELIZABETH NOLAN: I don't know the answer to that.
JOANNE STUBBE: OK.
Because ATP analogues have wide--
you guys have already seen ADPNP or ADPCH2P They really
have very different properties when you study these, the ATPs.
So if these-- and probably they don't have ATP because they
probably--
ELIZABETH NOLAN: Right.
They want to get a stable--
JOANNE STUBBE: So anyhow, that's something to keep
in the back of your mind.
ELIZABETH NOLAN: So just is this an artifact is what
JoAnne's suggesting from use of a non-hydrolysable ATP
analogue.
JOANNE STUBBE: And there's many examples
of that in the literature.
Everybody uses it.
It's just something you need to keep in the back of your mind.
That's the best we can do.
ELIZABETH NOLAN: So next week, someone
should ask during recitation there, for that.
So what about the mechanical work?
How this is often depicted, in terms of grabbing and pulling
on a polypeptide substrate, is via these rigid bodies.
And we're not going to go into details about this,
but just to describe the typical cartoon picture effectively,
imagine we have some polypeptide that needs to enter
the degradation chamber.
So those pore loops we heard about that
are involved in substrate binding
are in the large domain of ClpX.
So here we have one large domain,
and then we can have the small domain of the adjacent subunit
here.
And just imagine here we have another large domain
with its pore loop.
And then we'd have the adjacent subunit here.
So effectively, it's thought that these pore loops
in the large domains grip the substrate and help
drag the substrate to allow for translocation
into the degradation chamber.
So this would be going to the chamber, that direction
here for that.
So somehow the ATP binding and hydrolysis
is allowing this to occur--
so to ClpP here for that.
So next week in recitation, you're
going to have a real treat because an expert, Reuben, will
be discussing some single molecule methods that
have been applied to studying this degradation chamber.
So bring your questions to him because he really
knows what is state of the field right now for this.
So we've talked a lot about how the substrate needs to get in.
We have the SSRI tag.
We have all of this ATP consumption unfolding,
translocation by ClpX.
And then we talked about the serine protease mechanism
in terms of how peptides are degraded in the chamber.
So then the final question just going to touch upon is,
how does the polypeptide that's been degraded
get out of the chamber?
So ClpXP will give products that are
7 to 8 amino acids in length, so short polypeptides.
So how are they released?
And we can think about two possibilities
for how these polypeptides are released.
One is that they're released through the axial pores.
So somehow those pores that allow polypeptide substrate
to go in also allow product fragments to go out.
And then the second option is that there's release
through transient side pores between the ClpP 7-mers.
So effectively, if we imagine coming back to our ClpP,
we have a 7-mer--
back-to-back 7-mers, do the fragments
come out, say, of the hole?
Or somehow do they come out from this region here?
To the best of my knowledge, this is a bit unclear,
and I don't think they're mutually exclusive.
So questions have come up.
If they're to come out of an axial pore,
does that mean ClpX has to be dissociated?
In terms of this equator region, there
are structures showing that this degradation
chamber can breathe.
And there's a picture of that in the posted notes from Friday
where you can see opening here.
And there has been some experiments
done where people have put cysteines in this region
by site-directed mutagenesis.
So you can imagine, for instance,
if you have a cysteine here and a cysteine here,
and you oxidize to form a disulfide such that those two
7-mers are locked together.
You can ask, if we load the chamber with small polypeptides
and we have these effectively cross-linked by disulfides,
can the polypeptides get out?
And then if we reduce this to have them no longer attached
to one another, do those polypeptides
stay put or not there?
Those experiments gave some evidence
for release of peptides through this region here,
but there's also evidence for release of peptides
through the pore.
And in terms of cartoon depictions.
In the lecture notes, if you take a close look,
you'll see that both come out there.
So I'd say if you're curious about that,
you can read some of the literature
and come to some own conclusions.
One last point on the Clp system before we move on to module 4,
you should just be aware that there's other Clp family
members.
So not only ClpX and P. And so in the Clp system--
actually, I'm going to make one other point after this
too, about degradation chambers.
So there are players ClpA, ClpB, in addition to ClpX.
So these are all three different AAA+ ATPases.
And you've actually encountered ClpB last week.
So this is HSP 100, which came up in question 2 on the exam
there by another name.
And then in addition to ClpP, there's
also ClpS and some other players here for that.
They each have their own personality within protein
quality control here for that.
And then we've only looked at this degradation chamber
from bacteria.
You might want to ask the question,
what happens in other organisms?
And the answer is that the complexity varies and systems
become tremendously more complex as you move from bacteria
into eukaryotes there.
And so if we consider the different degradation chambers,
what do we see?
So we find these proteasomes in all forms of life.
And as I just said, the level of complexity
varies depending on the organism.
And so what we've seen with ClpP is the most simple system
where we have two rings that have only one type of subunit.
So just say E. coli.
One type of subunit.
What happens if we go to archae?
We find that we have four rings, each of which is 7-mer.
And these four rings include two different types of subunits.
So I'll call these alpha and beta.
So what we find is that there's a 7-mer of 7 alpha subunits,
then 7-mers that have 7 beta subunits, and here
a 7-mer with alpha.
So we see two types of subunit and four rings.
So then what about yeast?
Tremendously complex.
So we have this architecture again
of four rings, an organized alpha, beta, beta, alpha.
But what we find in this case is that in each of these--
I'm not going to draw it like that, but each of these
have seven different subunits.
There's a depiction of this in the notes.
So just imagine-- how does this get assembled?
I have no clue.
But somehow each of these heptamers
has to be assembled with seven different subunits.
And then they're put together in this series of four rings.
And then as you'll see after spring break in JoAnne's
section, the eukaryotic proteasome
has this 19S regulatory particle that's
involved in recognizing condemned proteins that
have polyubiquitin chains.
And compared to the ClpX ATPase, it's much, much more complex.
So there's many different proteins
that constitute this necessary part of the machine.
But there is a hexamer, ATPase hexamer, within there
to facilitate translocation of the polypeptide
into the degradation chamber.
So some of this will come back again
in the latter half of the class.
So with that, we're going to close on degradation and move
into module 4, which is focused on macromolecular machines that
are involved in the biosynthesis of natural products,
specifically polyketides and nonribosomal peptides.
And so we're completely taking a loop back
to thinking about a biological polymerization,
like what we were thinking with the ribosome
from the process of breaking down a polypeptide.
And so where are we going?
We can think about assembly lines,
although this is a helpful way on the board
to think about these systems.
But it's not really what they look like.
And you'll learn about that in recitation this week.
Yeah?
AUDIENCE: Could you explain the interaction
between ATP and the hinge area?
ELIZABETH NOLAN: OK.
So the ATP binding site is just rewinding here
in that hinge region.
And there's going to be conformational change
in the hinge with ATP binding and hydrolysis there.
And that's sufficient in terms of the level of detail
for this.
But the main thing to keep in mind, each subunit binds ATP.
But on the basis of the information gathered
with the caveats JoAnne brought up,
different subunits bind ATP at different times in the cycle.
AUDIENCE: OK.
Thank you.
ELIZABETH NOLAN: And changes in this subunit,
conformational changes that result from that,
can be translated to the next door subunit here.
AUDIENCE: OK.
ELIZABETH NOLAN: OK.
So where are we going?
By a week from now, you should have a good handle
on how to think about the biosynthesis of structures
like erythromycin, of penicillin.
These are products of assembly lines.
And so where we'll go is with a brief overview of fatty acid
biosynthesis and then look into polyketide synthase
and nonribosomal peptide synthetase assembly lines here.
And then some case studies.
So on the topic of ATP, where we just went back to with ClpX,
just taking a look here, what do you
know about these systems in ATP by the names?
This is just a little language use and definition.
So there's a subtle difference here.
What's the difference?
AUDIENCE: Synthase versus synthetase?
ELIZABETH NOLAN: Yeah.
And what does that tell you right off the bat?
About ATP.
So it's a subtlety, right?
Synthase is a general term.
Synthetase indicates ATP is involved.
So as we'll see, these nonribosomal peptide
synthetases employ ATP.
And we're going to see chemistry very similar to what
you saw with the aminoacyl-tRNA synthetases
in terms of activating amino acid monomers.
But in this case, the machine is forming a nonribosomal peptide
rather than a ribosomal peptide here.
If you are not familiar with fatty acid biosynthesis,
I highly encourage you to go do some review,
either from your 5.07 notes last term if you were in the class
or from a biochemistry book.
And there'll be some additional slides of overview information
posted online.
So we'll just touch upon it today but not
go into tremendous detail here.
So what are our questions for this module?
I think for most everyone in the room,
this module will contain the most new information
from the standpoint of a new system
compared to what we've talked about so far.
So what are polyketides and how are these molecules
biosynthesized by polyketide synthases?
What are nonribosomal peptides and how are they
made by these machines called nonribosomal peptide
synthetases?
And what we're going to look at is the assembly line
organization, so effectively the organization of domains that
provide these linear polymers.
So what is the assembly line organization and logic for PKS?
And likewise for NRPS.
And then we can ask, how can a given assembly
line for a given PKS or NRPS natural product
be basically predicted from the structure
of the natural product?
So you should be able to work back and forth in terms
of looking at a structure and coming up
with a biosynthetic prediction and also seeing
biosynthetic machinery and getting a sense as to what
that small molecule metabolite's backbone might look like.
How are these studied experimentally?
And we'll look at the biosynthesis
of a molecule called enterobactin as a case study.
And so one thing I'll just point out right now
is that these synthases and synthetases do not
look like an assembly line.
And we'll draw domains in a linear order which really
facilitates thinking about the chemistry,
but the structures are not just a line of domains or proteins
next door to one another.
And this week in recitation, you'll
get to see some cryo-EM studies on fatty acid synthase and
related machines there which will give you
a sense of their dynamics.
So just a review.
If we think about template-dependent
polymerizations in biology, we're
all familiar with DNA replication, transcription,
and translation.
And what you'll see in this unit is
that these template-driven polymerizations
occur in the biosynthesis of natural products here.
And effectively, these assembly lines, in a way,
provide this template.
So they're small molecules being biosynthesized
by microbes using some pretty amazing machinery.
So when we think about template-driven
polymerizations, we think about an initiation process,
elongation process, and termination.
We saw that with a translation cycle.
And we'll see the same type of systems here.
So what does some of these structures look like?
Here are just some examples on the top
of polyketides, two examples.
They look very different at first glance, and they are.
So we have tetracycline.
We have four fused 6-membered rings.
It's an aromatic polyketide, an antibiotic.
We have this erythromycin here, which is a macrolide.
We encountered macrolines in the translation section
because they bind the ribosome, another type of antibiotic.
If we look at some nonribosomal peptides, all of that
can be used clinically.
We see the penicillins.
So we have a 4 or 5 fused ring system here, a beta-lactam.
This comes from three amino acid building blocks initially.
We have vancomycin.
This is an antibiotic of last resort.
And this structure looks really quite complicated,
but what we'll see is that it's based on seven
proteogenic amino acids.
So it's the 7-mer peptide backbone
that gives rise to this structure here for that.
And then we see there's some sugars,
so these can be put on by other enzymes here.
So on top we see a lot of ketones and OH groups.
Those are good hints that maybe polyketide logic is being used.
Here we see a number of peptide bonds,
amide bonds, a good indicator of NRPS at play.
And here, just to point out, these systems get very complex.
And there's natural products out there
that are biosynthesized from a combination
of polyketide synthase logic and nonribosomal peptide synthetase
logic here.
These include molecules like yersiniabactin.
This is an iron chelator produced by Yersinia pestis,
and some pathogenic E. coli, this immunosuppressant
rapamycin as examples.
So as we move forward, I put a lot
of structures of small molecule metabolites in the slides.
You can go back and use them as a way
to study and try to make predictions
about what is the machinery at play,
for instance, to give all of these heterocycles?
How are those made?
We'll see the assembly line does that.
So what organisms produce these molecules?
Largely, bacteria and fungi.
And there are some correlations out there, I'll just point out,
related to genome size and the number
of metabolites being made.
So bioinformatics guides a lot of current studies
of the biosynthesis of these types of molecules.
So you can imagine that you sequence a genome.
You have some information about gene clusters.
So these are groups of genes where
the proteins work together to biosynthesize the molecule.
And often, the genes that encode proteins in these metabolites
are clustered.
And so bioinformatics approaches can help find these.
What's found is that for bacteria, some phyla
are more prolific producers of these molecules than others.
And what's been shown in a general way
is that organisms with small genomes--
so something like E. coli--
produce fewer of these metabolites.
That's not to say none.
So enterobactin, which we'll look at for a case study,
is made by E. coli.
But they don't make as many.
And effectively, organisms with larger genomes produce more.
And so here is just a correlation
between the number of genes.
And the genome size of the organism
where they see around 3 Mb, there's a switch here.
Often, these molecules-- yeah?
AUDIENCE: Is there any hypotheses
about an evolutionary driving factor
for the development of this machinery
and why it correlates to genome size?
ELIZABETH NOLAN: If there is, I don't know.
I don't think about evolution very well, quite frankly.
What is thought is that many of these molecules
are thought to be involved in defense
and that an organism with a smaller genome size
uses other strategies.
And so for instance, E. coli, which
I cited as a small genome, will use
a number of ribosomal peptides as defense molecules
that get post-translationally modified after the fact.
But why that organism chooses to do that versus say something
like Streptomyces that produces many, many different natural
products, I'm not sure about that.
So let's look at an example of a gene cluster,
just so you get a sense of how much machinery
is required to do the full biosynthesis of a molecule.
So this is for a nonribosomal peptide shown here.
It has some structural similarities
to the vancomycin we saw on a prior slide,
and it is a member of the vancomycin family.
So this gene cluster for the biosynthesis of this metabolite
contains 30 different genes and is depicted here.
So each one of these arrows indicates an open reading
frame.
So each one begins with a start, ends with a stop codon.
And it's assumed to be the coding sequence of the gene.
And so what is encoded in these 30 genes?
Well, first there are the genes for what
we call the assembly line.
And if it isn't clear what assembly line means,
as we move forward through this week, it will be.
So there's genes required to make the 7-mer polypeptide
backbone.
There's genes required for modification of the backbone.
So how do these sugars get attached, for instance?
Those are going to be some tailoring enzymes.
And then if you take a close look,
there's a number of non-proteinogenic amino acids
in this molecule, and that means they
have to come from somewhere.
And so this gene cluster also includes
genes that are required for the biosynthesis of those monomers.
So there's a lot of effort going in to making
this molecule by some organism.
And so presumably, under some set of conditions,
it's important.
So moving towards the chemistry, with that background in hand,
what are some points to make?
So what we'll learn and see is that the assembly lines that
produce the polyketides and nonribosomal peptides
are macromolecular machines.
So there's dedicated macromolecular machines
for the biosynthesis of these secondary metabolites.
And so what are secondary metabolites
versus a primary metabolite?
So what's a primary metabolite?
AUDIENCE: I'm not even totally sure how to define metabolites.
Isn't metabolites what goes in?
Or what comes out?
ELIZABETH NOLAN: Rebecca?
AUDIENCE: Or easily produced directly from the materials
the cell's consuming?
ELIZABETH NOLAN: So presumably, the cell
needs to get materials to biosynthesize
the secondary metabolites too, right?
Somewhere, these amino acid monomers
or the monomers that are used for polyketide synthetase
need to--
they'd have to come from somewhere, right?
So are primary metabolites important for growth?
AUDIENCE: Yes.
ELIZABETH NOLAN: Yes.
Development?
Reproduction?
AUDIENCE: Yes.
ELIZABETH NOLAN: Yeah, right.
Under normal conditions, right?
We're in trouble if we don't have our primary metabolites
there, whether they're ingested or biosynthesized.
What about a secondary metabolite?
Just taking that--
AUDIENCE: I'm guessing it's not necessary.
AUDIENCE: --something we can make from primary metabolites?
ELIZABETH NOLAN: No.
Well, you can.
You can.
So a secondary--
AUDIENCE: --necessary?
ELIZABETH NOLAN: Yeah.
A secondary metabolite is not required for normal growth,
development, reproduction.
So for some reason, under some circumstances of need,
these secondary metabolites get produced.
So for some of these antibiotic molecules, maybe
the organism needs to defend itself.
In the case of enterobactin or yersiniabactin,
maybe that organism needs iron.
And so it's producing a molecule that
will help it obtain that there.
So what is going on?
We've seen some pretty complex molecules.
What we're going to see is that these assembly lines convert
simple acid monomers, if it's a polyketide synthase
or amino acid monomers for a nonribosomal peptide
synthetase, into linear polymers.
So we're going to look at template-driven polymerizations
that initially give linear polymers.
And in the case of PKS, this is very similar
to fatty acid biosynthesis.
What we see is that the assembly lines
allow for iterative additions of malonyl and methylmalonyl
units.
And they catalyze carbon-carbon bond formations.
In the case of nonribosomal peptide synthetases,
what we'll see is that these allow for condensations
of amino acids to form peptide bonds
and effectively form nonribosomal polypeptides.
So polypeptide synthesis without the ribosome.
So even though the PKS and NRPS are forming a different type
of bond and that requires different chemistry, what
we'll see is that they use very similar logic.
And just getting the logic sorted out initially
makes life much easier down the road.
So take some time to look over the depictions in the notes
outside of class as we go forward.
So these assembly lines use acyl or aminoacyl thioesters
as the activated monomer units.
So then how do we get from this linear polypeptide
to some more complex structure?
The short message on that is that the, quote, "polymers"
that are produced--
and they may be short, right?
We just saw-- they are short, 7 amino acids for vancomycin.
They can undergo further elaboration
to give these complex structures.
So there can be tailoring enzymes
that work on the products of the assembly line.
Or there can be domains in the assembly line that
give additional activities that allow for methylation
or cyclization here.
So we can think about fatty acid synthase as a paradigm here.
And so if we think about fatty acid
biosynthesis making some molecule like this oil here,
just as brief overview in the last few minutes of class.
Fatty acids are synthesized by FAS.
And what happens is that there's elongation
by one unit at a time.
And each unit provides two carbons.
So there's two carbon atoms per elongation.
And so hopefully you're all familiar with two ways
to form a carbon-carbon bond, at least related
to biochemistry, one of which is Claisen condensations.
So Claisen condensations allow for carbon-carbon bond
formation and join the units.
To keep in mind, the monomers are always
thioesters, not oxoesters.
And for fatty acid biosynthesis, the two monomer units
are shown here.
So we have a starter and an extender, acetyl CoA or malonyl
CoA here.
And here we have coenzyme A.
So just as a brief review, if we think about these monomer
units--
so here we have acetyl CoA.
So what can we say about this guy here, in this thioester?
So is this acidic or not?
Compared to an oxoester.
How many of you have heard about fatty acid biosynthesis?
AUDIENCE: [INAUDIBLE]
ELIZABETH NOLAN: So why are thioesters used and not
oxoesters?
AUDIENCE: [INAUDIBLE] use the other end?
ELIZABETH NOLAN: OK.
So we'll go into a little more detail on Friday
to make sure the chemistry is straight here
because I'm not certain it is.
So--
AUDIENCE: Is oxoester referring to not that [INAUDIBLE]----
ELIZABETH NOLAN: OK.
So for Friday, think about a thioester versus an oxoester,
and how do properties differ?
And why might we want to be using thioesters?
And also review the Claisen condensation
because that's the chemistry that's
going to be happening to form the carbon-carbon bonds
in the fatty acid synthase and in the polyketide synthases.
And what we're going to see is that the monomers in each case,
they're tethered as thioesters.
So why is that?
And I will turn around and point at somebody,
and you can let us know.
Are you excited?
OK.
So you're off the hook for Wednesday.
I need to be out of town, and I'll see you on Friday.