Hi, I'm Art Horwich, from Yale School of Medicine.
And I want to describe another little segment
of a chaperonin experiment
that we carried out that came at the issue
of where proteins might be folded
by chaperonin ring structures.
And so, the question is:
where are polypeptides actually folded by these machines?
Are they folded inside the machine
or are they folded outside in solution,
after release from them?
This was a murky question at one point,
with the field fairly divided about whether
folding could occur inside chaperonins
or whether chaperonins simply were used to unfold proteins
that had undergone misfolding
and then release them back into free solution,
where they could productively fold,
potentially.
So, the people who worked on this problem
were Wayne Fenton, Corrine Hohl, and Jonathan Weissman
as the principals.
And so here is where we sat
when we first had an unliganded structure of GroEL.
We imposed the native structure
of one of our favorite substrate proteins for the chaperonin,
namely the sulfurtransferase called Rhodanese, at 33 kD protein,
upon the unliganded structure of GroEL.
And what you can see is that
there's a pretty terrible steric clash here,
where the apical domains
-- sorry, the apical domains of GroEL --
are born upon by Rhodanese,
even in its native state.
Of course, in a non-native state,
we'd expect the protein to be somewhat expanded,
so the steric clash with the apical domains
would be even worse than what you see here.
And so this suggested that maybe folding
could not occur inside the machine,
but our thinking about that was rapidly changed
by our long-time collaborator Helen Saibil,
shown here on one of the few sunny days in London,
sitting in her backyard garden reading a book,
or Nature, or some other journal.
In any case, what Helen showed,
from single particle reconstructions
using cryo-EM or negative stain imaging
with the results being roughly the same in both cases,
was that when GroES binds to a GroEL ring,
and remember what you just saw
was unliganded GroEL,
when GroES binds,
now the apical domains of the bound ring open up
and there's a space underneath GroES, shown here,
that is fairly considerable, substantially larger
than the volume of the cavity
of an unliganded GroEL ring.
And the presence of that space
excited us hugely, because suddenly it seemed possible
that a polypeptide could actually
be fit into that space,
and maybe folding could even take place
in that location.
So Jonathan Weissman, who was a postdoc in the lab at that time,
carried out an order-of-addition proteolysis experiment.
So, he used two different orders of addition.
As shown at the top here,
he added GroES first to GroEL,
followed by polypeptide,
and then followed by treatment
with the protease Proteinase K.
And so, as you can see here,
the topology is such that,
if GroES binds asymmetrically,
that will more of less encapsulate this ring,
and polypeptide is unable to bind to that ring.
It'll rather bind in trans to the opposite ring,
and then upon addition of Proteinase K,
will become completely digested.
And that is in fact what he observed.
And the prediction was that
if you reverse the order of addition
such that polypeptide is added first,
then followed by GroES,
if GroES comes on randomly,
either to the same ring as polypeptide,
or to the opposite ring of polypeptide,
two different results would obtain.
When it comes on the same ring,
the polypeptide should be protected from Proteinase K.
When it comes on the opposite ring,
polypeptide is really effectively bound in trans,
and is susceptible then to Proteinase K digestion.
And indeed, when he carried out this experiment,
he observed that roughly 50%
of the substrate protein that was put in
at the original step over here
was protected from Proteinase K digestion,
and he used different substrate proteins
including Rhodanese, OTC,
and one or two other proteins.
So to confirm, in fact, the topology of
-- a cis topology in particular,
namely that a polypeptide could be bound, or housed,
underneath a GroES inside of a GroEL ring --
he carried out a hit-and-run crosslinker experiment.
So, this availed of a very cute photoactivatable crosslinker
that could be iodinated,
the crosslinker is called APDP,
I won't read out the whole name,
but the point is that the guts of this crosslinker
are a salicylamide group here.
So on the ring of the salicyl group,
one could iodinate to make this crosslinker hot,
and it also had an azido group
that is a photoactivatable crosslinker.
At the other end of the molecule is essentially
-- if I can point it out correctly here
, is a disulfide hooked to a pyridyl leaving group
that allows one to place this crosslinker,
through a cysteine,
onto a substrate protein.
So, for example, Rhodanese, one of our favorite substrates,
has I believe four cysteines on it.
And so you could efficiently put this crosslinker on Rhodanese,
and now bind Rhodanese to GroEL,
here the crosslinker is symbolized by this star,
and one could then shine light on this complex,
and that would photocrosslink
through the azide group.
That ring would now become
covalently attached to the proximate GroEL ring
that is near the polypeptide chain,
and one could then reduce with DTT
and the polypeptide would effectively leave
such that only the iodinated group
is left on the GroEL ring
that was near to the polypeptide.
Okay, so that's the photocrosslinker,
and the experiment that was essentially employed.
And here is the results of a key
order-of-addition that confirms the cis topology.
So once again, the order
at the level of the reaction
is to add our substrate protein, OTC,
or in this experiment Rhodanese,
then add GroES,
now Proteinase K to get rid of any polypeptide
that's in a trans ring,
and now shine light to essentially photocrosslink,
and then look to see where bands
would appear on an SDS gel
upon denaturation of these complexes.
And so if I can get sufficiently
out of the frame of this experiment,
here you see these orders of addition:
so substrate, with a photocrosslinker,
is added in the first step to the left,
then GroES is added to
either encapsulate in cis or bind in trans,
then Proteinase K is added,
and you can see that the cis-localized polypeptide
is protected here,
but digested here,
and then shine light to photocrosslink,
as shown here.
And so now the polypeptide is
photocrosslinked in cis,
and if it's photocrosslinked in cis,
the Proteinase K will have cleaved the tails
off of the subunits that are in trans,
but won't touch the cis subunits
because it's protected by the presence of GroES,
and thus one would see essentially
an uncut polypeptide.
And that is what you see when you come
all the way over there and look on the SDS gel,
is that most of the material is, indeed, uncut.
It's present in the cis complex.
And so this hit-and-run crosslinking experiment,
placing a photocrosslinker that could be
essentially, effectively, reduced off of
bound Rhodanese after photocrosslinking,
allowed us to see that
a cis topology was in fact possible.
And so, in a next step,
we asked whether cis or trans complexes
were capable, in either case,
of being productive of the native state of a polypeptide.
And so Wayne Fenton in the group
made either trans complexes with OTC
or cis complexes.
So in the case of trans complexes,
he bound GroES first in the presence of ADP,
then bound OTC in trans.
In the case of cis complexes,
he bound OTC first, then added GroES
and magnesium-ADP to encapsulate,
but also to get rid of anything,
any OTC that was bound in trans,
he treated with Proteinase K,
and now he had selective populations
of either trans or cis OTC complexes.
And now he carried out
activation of folding using ATP,
added for only brief periods of time before
treatment with apyrase to digest the ATP,
and I'm going to describe how he did this experiment,
in fact, under single-turnover conditions
that allowed us to establish that, in fact,
folding starts at cis complexes
and not at trans ones.
The trans complexes are not productive.
It was an interesting argument in the lab,
nobody really had a clear idea
what was going to work, but it turns out that cis,
somewhat to our surprise,
turned out to be productive of the native state of OTC.
So, now I have to back up
to describe the single-turnover aspect of this experiment.
So, it availed of the ability
to produce GroEL as a single-ring version.
And so here you're looking at the interface
between the top GroEL ring
and the bottom GroEL ring, or at one portion of it.
So shown here is one subunit on top,
of the top ring of GroEL,
but it is actually in contact with two adjacent subunits
in the bottom ring.
To the left you see a Glu434
that is from one subunit of the bottom ring
making a sort of nearby contact
to Glu434 from the relevant subunit of the top ring.
There's actually a two-fold symmetry axis
that would go right into the board at that position.
And also, at the right-hand contact here,
there's another sort of two-fold symmetry axis
that goes -- I don't know if I can point --
about right here, into the board.
And you see that there is the two-fold
arrangement of a series of residues
that include Arg452, Glu461,
Val464, and Ser463.
The bottom line is when all of these residues,
these four residues,
were simultaneously changed to alanine,
now what one saw was a single-ring version of GroEL
produced in E. coli when that mutant construct was expressed.
And so a single-ring version of GroEL
was of considerable value to us
because, for the experiment that I'm going to describe to you,
Wayne's experiment,
in a little bit more detail,
the single-ring version of GroEL
could serve as a GroES trap.
That is, it could bind GroES,
but since it had no opposite ring,
it could never release it.
And so in Wayne's experiment, the cis or trans test,
what he did was to add SR1 as a trap for GroES,
magnesium-ATP for short periods of time,
and then apyrase.
So, the reaction starts here,
or in cis over here,
and it can really only go one route,
because there's a huge excess of the trap,
GroES is only released once
before it's completely trapped by these molecules
and the polypeptide can go where it goes.
So, as it turns out, the cis complexes
are productive and produced the OTC trimer.
By contrast, the trans complexes
do not produce any OTC enzymatic activity,
probably most of the OTC molecules
simply rebind to GroEL and go nowhere,
they're just bound.
I doubt whether they're very stable free in solution,
they would probably tend to aggregate under those conditions.
But certainly there's no OTC activity
produced by a trans complex under these conditions.
Okay, so with that in mind,
we could say that polypeptide chain folding
commences at a cis ternary complex.
The questions was:
could it go all the way to completion
inside of a cis ternary complex?
And for that experiment, once again
we could use this SR1 version of GroEL,
this single-ring version where
we had shaved off the residues, with mutations,
that lie at the equatorial interface,
and thus produce obligatorily a single-ring version of GroEL.
So Corrinne Hohl was a visiting student
from Duke University one summer,
and she and Jonathan carried out this experiment.
So basically, what they did was to take SR1,
add Rhodanese to it to form a binary complex
of Rhodanese and SR1,
after all the apical domains of the open ring version
of SR1 are perfectly capable of binding non-native polypeptide,
and now they added ATP and GroES
to that complex.
And so that formed what is effectively
a stable cis ternary folding chamber,
unable to be ejected because there is no opposite ring
with which ATP binding could eject the cis ligands.
And so therefore, they could ask whether Rhodanese
could go all the way to the native state
inside of this 400 kD complex
that they had formed.
And so on the top panel here is just
the total recovery of Rhodanese enzymatic activity
at this reaction free in solution.
So, they've just analyzed total Rhodanese activity
of the reaction mixture,
and what they see is that the recovery of Rhodanese
at SR1/GroES/ATP, shown on top here,
is the same as that at a wild type reaction.
And in fact, as evidence that
the refolding is occurring inside of this complex,
what they did at the various time points
in a second experiment
was to carry out gel filtration
and isolate the 400 kD fraction
off the gel filtration column
from each of these time points down here,
and assay for Rhodanese
the material that came off the gel filtration column.
And what they observed was that they saw
the exact kinetics of recovery
with the gel filtered material as they observed
from looking at the total reaction, as shown above.
And so this demonstrated that polypeptide chain folding
could go all the way to completion
inside of a cis ternary folding chamber.
So the conclusion is that productive folding by GroEL
occurs in a GroES-encapsulated cis folding chamber.
And I would just point out that
the characteristics of this chamber
are to be very hydrophilic,
in fact quite electrostatic with a little bit of an excess of negative charge,
but we think that all of this charge presents, essentially,
a no-stick surface that effectively allows
the polypeptide chain to pursue its way down the energy landscape
to the native state without real interference.
Almost as if it were at infinite dilution.
So our supposition is that the walls of GroEL
really don't carry any steric information,
even though there are collisions almost certainly
of non-native proteins with those walls,
but rather provide an encapsulated chamber
where aggregation of multiple molecules
simply cannot occur
and the isolated polypeptide chain
has time to try out different conformations
and ultimately reach the native conformation
at the energetic minimum.