Hi, I'm Art Horwich from Yale School of Medicine.
I want to describe one of the experiments
that we've carried out over the years
to try to understand how chaperonins
mediate protein folding.
And today, in this particular segment,
I want discuss experiments that we carried out
that get at the role of ATP,
essential for chaperonin-mediated folding,
the binding and hydrolysis of ATP by the GroEL machine,
and how that drives the chaperonin cycle.
So, the questions that I want to raise
are shown here.
What does ATP do is the first
sort of fundamental question.
There was some history coming into our experiments;
it was known from early experiments
that ATP binding was required
in the seven subunits of a ring
to recruit GroES to a ring,
to bind it and encapsulate that particular ring.
I was also known from early on,
from work of Yifrach and Horovitz at the Weizmann institute,
that there is binding of ATP cooperatively
within a GroEL ring,
but anti-, or negatively-cooperative between rings
such that, at any given time,
a GroEL is occupied with ATP on one ring,
but it does not have nucleotide
present on the opposite ring.
Thus, GroES can only bind
to a ring that is occupied with ATP,
and this produces an inherent asymmetry
of the chaperonin/co-chaperonin complex,
with GroES typically binding to only one GroEL ring at a time.
So I'd like also to take up the issue
of when is binding required
and when is hydrolysis required?
It turns out that there are distinct actions
of binding and hydrolysis.
And last but not least, I want to acknowledge
the experimentalists who were involved with this work:
Hays Rye, who was a postdoc in the lab at that point,
Charu Chaudhry, a student shared with the Paul Sigler group,
and Steve Burston, who was a postdoc in the lab.
So, first I'll talk about the GroES-bound ring
and the behavior of ATP in this
so-called cis ring.
And so I should first point out
a little bit of the geometry
and architecture of a cis complex,
an asymmetric GroEL/GroES complex.
So here you see GroES bound
to the top of a GroEL ring
and the point is that it's a nucleotide-occupied ring,
and the point further is that
at the nucleotide pocket there is a stereochemistry
that gives us some pointers
on where mutagenesis
and structure/function studies would be most informative.
So it turns out that when
ATP is bound in the pocket,
and here this is actually ADP-aluminum fluoride complex
that was co-crystallized with GroEL/GroES
and produces an asymmetric complex,
here is where you see, effectively,
the gamma phosphate, with is an AlF3 complex.
You see the magnesium cation
that is present also in the ADP state of GroEL,
but you see now here also,
crucially, a residue from the intermediate domain of GroEL
that is essentially swung down into the nucleotide pocket.
The residue is Asp398, aspartate 398,
and basically this acidic residue is going to activate a water,
which is not shown very well in electron density,
and so I'll just point to where it would be with my finger.
Between Asp398 and Asp52
there would be the activation of that water
to hydrolyze the gamma phosphate,
more or less, off of an ATP.
And so if one changes Asp398 to an alanine,
a GroEL can very efficiently bind ATP,
with more or less the same affinity
as the wild type molecule,
but the mutant molecule fails to hydrolyze, as excepted,
ATP with a reduced rate,
reduced to the level of about 2% of wild type.
So effectively, hydrolysis is
substantially blocked or slowed
to a very slow rate.
So what that stereochemistry in mind,
we've used the Asp398 mutation
to assess functions
both in the cis ring and trans ring of GroEL,
and so the first observation we could make
is that ATP binding alone,
in the absence of hydrolysis
due to this D398A mutation,
is sufficient to trigger productive folding.
And so this was an experiment carried out
with a single-ring version of GroEL
that I'll describe in a little bit more detail
in a few slides.
But it essentially has mutations
at the equatorial aspect of a GroEL ring
that prevent the two rings from normally associating.
And so this single-ring version of GroEL
that also contains a D398A mutation
can effectively bind ATP
and thus move its apical domains and bind GroES,
and effectively form a folding chamber
in which a polypeptide could be released and folded,
in the absence here, because of this mutation,
of ATP hydrolysis.
And so ATP binding to SR398
is sufficient to trigger productive folding
of a pyrenated Rubisco,
but also of substrates like Rhodanese
that are monomeric substrates
that can be assayed directly for their recovery
of enzyme activity,
while they're in this 400 kD complex.
And so these items of data
indicate that ATP binding,
in the absence of hydrolysis,
is sufficient to trigger productive folding.
So this, I would say, its the minimal folding-active version
of the GroEL/GroES chaperonin system.
And so here you're seeing just a little bit of this data
in real time from the Rubisco experiment.
What we're watching here is fluorescence anisotropy
as shown in this top panel.
And you can see that either wild type GroES/ATP,
or an SR1-GroES/ATP,
trigger, upon addition of ATP,
a rapid drop in the anisotropy of a substrate Rubisco,
followed by a slow rise.
So that corresponds, that slow rise,
to the recovery of Rubisco enzyme activity
in a complete folding reaction.
Note in the bottom panels here, though,
that in the presence of ADP or the non-hydrolyzable analog AMPNP,
no such fluorescence changes occur,
and to our understanding,
the polypeptide remains stuck to the apical domains
of GroEL, with release unable to be triggered
absent the gamma phosphate of ATP.
So, and here just to add to this,
you see SR398 that I mentioned a couple slides ago
-- the single-ring version that cannot hydrolyze ATP,
despite being able to bind it efficiently --
it binds GroES efficiently
and it forms an encapsulated chamber, and indeed SR398
produces a fluorescent anisotropy change
that has the same rise that we see
with the wild type complex or with SR1,
that corresponds to refolding of Rubisco
inside the folding chamber.
And once again, the presence of non-hydrolyzable analog,
like AMPPNP,
does not support the kinds of fluorescent anisotropy changes,
or in the case of Rhodanese activity changes,
that correspond to production of the native state.
So the gamma phosphate of ATP, and ATP itself,
are crucial in the cis ring to triggering productive release
of polypeptide from the walls of the...
from the GroEL cavity walls
into what is a cis folding chamber.
Okay, so in fact we've been able to do experiments
where we add, effectively, the gamma phosphate of ATP
in a separate step.
So, one first forms an ADP cis ternary complex.
For example, using Rhodanese,
one would add Rhodanese to SR1,
then add ADP and GroES,
and this forms, in fact, an encapsulated complex.
But from earlier fluorescence anisotropy experiments,
the Rhodanese is stuck on the cavity wall:
it's never released into the folding chamber.
By contrast now,
if one adds to this stable complex,
aluminum fluoride metal complex over here,
effectively, all hell breaks loose:
the polypeptide is released from the cavity wall,
and we now see recovery of Rhodanese activity
at a rate that corresponds to folding inside SR1-GroES,
or at a wild type GroEL/GroES reaction.
So, effectively, the addition of that aluminum fluoride complex,
and I should say this can also be mimicked with beryllium fluoride,
which is a ground state analog
as opposed to the transition state analog
that aluminum fluoride is usually thought of,
either of those is capable of producing
a release of the substrate into this folding chamber,
where productive folding ensues
and the native state of Rhodanese is produced.
Okay, so now,
having dealt with the action of ATP in the cis ring,
I'd like to turn to its actions in the trans ring.
So, this is the ring
that is opposite the one bound by GroES.
So what does ATP do in that particular ring?
And so, to carry out these experiments,
Hays Rye and Jonathan Weissmann
and a number of people in the group,
used effectively a double-ring version of D398A GroEL.
So again, this is a mutation
that allows ATP to bind with normal affinity
to the seven subunits of a GroEL ring,
but minimizes the rate of hydrolysis
to 2% or less of wild type.
So, effectively, one could form
a cis ternary complex in which ATP
is bound to a GroEL ring,
to a substrate-bound ring that is,
and recruit GroES to encapsulate that substrate,
in this case it's GFP, so we can report on its refolding
easily enough by its recovery of fluorescence.
And so now we have a cis ternary complex
with GFP refolded over a period
of some minutes
inside the cis folding chamber of D398A.
One then carries out a series of steps
in which one removes any GFP
that was bound to the opposite ring
by treating with a protease, Proteinase K,
so that removes GFP such that
we only have GFP in the cis ring.
One then gel filters this complex
to remove any of the added excess ATP
that is not bound in this complex.
And then one incubates for various periods of time.
Now, we know that ATP hydrolyzes
very slowly in a D398A ring.
So we have to wait a while,
since it's 2% of the normal rate,
we have to wait a considerable while
before ATP in this ring will be hydrolyzed,
but the postulate is that once it hydrolyzes,
and this is according to the beautiful experiments
of Yifrach and Horovitz,
that once ATP hydrolyzes in this ring,
it will gate entry of ATP into the opposite ring.
And since that's a D398A ring,
that ATP will not hydrolyze on any short timescale.
It'll simply bind and do whatever it's going to do.
And so we could ask whether ATP binding
in that ring is sufficient to eject
all the ligands from the cis ring.
So there had been experiments
published in 1994 or so,
by George Lorimer and his coworkers,
that suggested that the action of ATP
-- they thought it was hydrolysis
but it turns out to really be binding --
the action of ATP in the trans ring
could eject GroES and the ligands from the cis ring.
And here was the further test of that model using D398A.
And so what we saw was that at short times
-- so this is a series of gel filtration runs
that you see laid out here --
at short times, you see that all of the GFP is present
-- excuse my pointing --
in the complex here,
that is the D398A complex.
This is free GFP that has been released from the complex.
What you see is that at a short time of 30 minutes,
there hasn't been sufficient hydrolysis in cis
to allow ATP to arrive in trans,
hence there's no release of GFP
that has refolded from the cis folding chamber.
And, at two hours, however,
the story is quite different.
Here, you see release of GFP, fairly efficiently,
from the cis folding chamber by the addition of ATP.
By contrast, non-hydrolyzable analogue and ADP itself
are unable to mediate, in trans,
the triggering of release of the ligands from the cis ring.
So we're talking about an allosteric reaction here
where ATP binds in trans
and sends a signaling across the ring/ring interface,
across a distance of about 40 or 50 Angstroms, to trigger
-- or more than that actually,
more like 80-100 Angstroms,
really, if we're talking about --
triggering the release of GroES,
polypeptide (here GFP),
but also of hydrolyzed ADP from the cis ring.
So it's an allosteric reaction that's driven
from the trans ring upon ATP binding.
So, the conclusion is that
binding to the trans ring, of ATP,
and release of the ligands,
requires hydrolysis in cis,
and in the case of D398A where hydrolysis is very slow,
you have to wait around for a couple hours.
In the normal situation,
one would wait around for about 10 seconds
while the cis ring hydrolyzes ATP to ADP
and gates the entry of ATP into the trans ring,
ejecting then the cis ligands.
So the overall conclusions are that ATP binding
really does major work in both the cis and trans rings.
In the cis ring, ATP binding
promotes the binding of GroES
and associated with that, the large structural changes
-- rigid-body movements --
of the cis ring apical and intermediate domains,
that basically contribute to ejecting polypeptide
into the folding chamber
where it commences to fold.
ATP binding in trans then,
gated by hydrolysis in cis,
triggers an allosteric ejection
of all of the cis ligands:
GroES, polypeptide, and ADP.
And so really the role of ATP hydrolysis here
is simply to advance the machine directionally.
So cis hydrolysis
gates entry into the trans ring of ATP,
which arrives at a rate of about 100/second.
It gates also the entry of polypeptide,
which binds more slowly at 5-10/second,
as we've published in recent experiments.
There have also been publications from Tony Clarke's group and others
measuring these rates of arrival.
And finally, GroES is the laggard,
the final ligand to arrive on an open ring,
with an arrival rate with about 1-2/second as a rate.
And so this assures efficiently encapsulation,
the fact that GroES arrives
as the slowest ligand to arrive,
it ensures ternary complex formation,
ejection of the substrate into the cis cavity,
and productive folding:
a new folding-active cis ring
on what had been the old trans ring.
So that is the sort of summary
of what I wanted to say about this system
and how ATP acts therein.