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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,