ATP-triggered conformational changes delineate substrate-binding and -folding mechanics of the GroEL chaperonin.

The chaperonin GroEL assists the folding of nascent or stress-denatured polypeptides by actions of binding and encapsulation. ATP binding initiates a series of conformational changes triggering the association of the cochaperonin GroES, followed by further large movements that eject the substrate polypeptide from hydrophobic binding sites into a GroES-capped, hydrophilic folding chamber. We used cryo-electron microscopy, statistical analysis, and flexible fitting to resolve a set of distinct GroEL-ATP conformations that can be ordered into a trajectory of domain rotation and elevation. The initial conformations are likely to be the ones that capture polypeptide substrate. Then the binding domains extend radially to separate from each other but maintain their binding surfaces facing the cavity, potentially exerting mechanical force upon kinetically trapped, misfolded substrates. The extended conformation also provides a potential docking site for GroES, to trigger the final, 100° domain rotation constituting the "power stroke" that ejects substrate into the folding chamber.

A small molecule inhibitor selective for a variant ATP-binding site of the chaperonin GroEL.

The chaperonin GroEL is a megadalton-sized molecular machine that plays an essential role in the bacterial cell assisting protein folding to the native state through actions requiring ATP binding and hydrolysis. A combination of medicinal chemistry and genetics has been employed to generate an orthogonal pair, a small molecule that selectively inhibits ATPase activity of a GroEL ATP-binding pocket variant. An initial screen of kinase-directed inhibitors identified an active pyrazolo-pyrimidine scaffold that was iteratively modified and screened against a collective of GroEL nucleotide pocket variants to identify a cyclopentyl carboxamide derivative, EC3016, that specifically inhibits ATPase activity and protein folding by the GroEL mutant, I493C, involving a side chain positioned near the base of ATP. This orthogonal pair will enable in vitro studies of the action of ATP in triggering activation of GroEL-mediated protein folding and might enable further studies of GroEL action in vivo. The approach originated for studying kinases by Shokat and his colleagues may thus also be used to study large macromolecular machines.

Requirement for binding multiple ATPs to convert a GroEL ring to the folding-active state.

Production of the folding-active state of a GroEL ring involves initial cooperative binding of ATP, recruiting GroES, followed by large rigid body movements that are associated with ejection of bound substrate protein into the encapsulated hydrophilic chamber where folding commences. Here, we have addressed how many of the 7 subunits of a GroEL ring are required to bind ATP to drive these events, by using mixed rings with different numbers of wild-type and variant subunits, the latter bearing a substitution in the nucleotide pocket that allows specific block of ATP binding and turnover by a pyrazolol pyrimidine inhibitor. We observed that at least 2 wild-type subunits were required to bind GroES. By contrast, the triggering of polypeptide release and folding required a minimum of 4 wild-type subunits, with the greatest extent of refolding observed when all 7 subunits were wild type. This is consistent with the requirement for a "power stroke" of forceful apical movement to eject polypeptide into the chamber.

Multiple states of a nucleotide-bound group 2 chaperonin.

Chaperonin action is controlled by cycles of nucleotide binding and hydrolysis. Here, we examine the effects of nucleotide binding on an archaeal group 2 chaperonin. In contrast to the ordered apo state of the group 1 chaperonin GroEL, the unliganded form of the homo-16-mer Methanococcus maripaludis group 2 chaperonin is very open and flexible, with intersubunit contacts only in the central double belt of equatorial domains. The intermediate and apical domains are free of contacts and deviate significantly from the overall 8-fold symmetry. Nucleotide binding results in three distinct, ordered 8-fold symmetric conformations--open, partially closed, and fully closed. The partially closed ring encloses a 40% larger volume than does the GroEL-GroES folding chamber, enabling it to encapsulate proteins up to 80 kDa, in contrast to the fully closed form, whose cavities are 20% smaller than those of the GroEL-GroES chamber.

Topologies of a substrate protein bound to the chaperonin GroEL.

The chaperonin GroEL assists polypeptide folding through sequential steps of binding nonnative protein in the central cavity of an open ring, via hydrophobic surfaces of its apical domains, followed by encapsulation in a hydrophilic cavity. To examine the binding state, we have classified a large data set of GroEL binary complexes with nonnative malate dehydrogenase (MDH), imaged by cryo-electron microscopy, to sort them into homogeneous subsets. The resulting electron density maps show MDH associated in several characteristic binding topologies either deep inside the cavity or at its inlet, contacting three to four consecutive GroEL apical domains. Consistent with visualization of bound polypeptide distributed over many parts of the central cavity, disulfide crosslinking could be carried out between a cysteine in a bound substrate protein and cysteines substituted anywhere inside GroEL. Finally, substrate binding induced adjustments in GroEL itself, observed mainly as clustering together of apical domains around sites of substrate binding.

Two families of chaperonin: physiology and mechanism.

Chaperonins are large ring assemblies that assist protein folding to the native state by binding nonnative proteins in their central cavities and then, upon binding ATP, release the substrate protein into a now-encapsulated cavity to fold productively. Two families of such components have been identified: type I in mitochondria, chloroplasts, and the bacterial cytosol, which rely on a detachable "lid" structure for encapsulation, and type II in archaea and the eukaryotic cytosol, which contain a built-in protrusion structure. We discuss here a number of issues under current study. What is the range of substrates acted on by the two classes of chaperonin, in particular by GroEL in the bacterial cytoplasm and CCT in the eukaryotic cytosol, and are all these substrates subject to encapsulation? What are the determinants for substrate binding by the type II chaperonins? And is the encapsulated chaperonin cavity a passive container that prevents aggregation, or could it be playing an active role in polypeptide folding?

Perturbed ATPase activity and not "close confinement" of substrate in the cis cavity affects rates of folding by tail-multiplied GroEL.

Folding of substrate proteins inside the sequestered and hydrophilic GroEL-GroES cis cavity favors production of the native state. Recent studies of GroEL molecules containing volume-occupying multiplications of the flexible C-terminal tail segments have been interpreted to indicate that close confinement of substrate proteins in the cavity optimizes the rate of folding: the rate of folding of a larger protein, Rubisco (51 kDa), was compromised by multiplication, whereas that of a smaller protein, rhodanese (33 kDa), was increased by tail duplication. Here, we report that this latter effect does not extend to the subunit of malate dehydrogenase (MDH), also 33 kDa. In addition, single-ring versions of tail-duplicated and triplicated molecules, comprising stable cis complexes, did not produce any acceleration of folding of rhodanese or MDH, nor did they show significant retardation of the folding of Rubisco. Tail quadruplication produced major reduction in recovery of native protein with both systems, the result of strongly reduced binding of all three substrates. When steady-state ATPase of the tail-multiplied double-ring GroELs was examined, it scaled directly with the number of tail segments, with more than double the normal ATPase rate upon tail triplication. As previously observed, disturbance of ATPase activity of the cycling double-ring system, and thus of "dwell time" for the folding protein in the cis cavity, produces effects on folding rates. We conclude that, within the limits of the approximately 10% decrease of cavity volume produced by tail triplication, there does not appear to be an effect of "close confinement" on folding in the cis cavity.

Global aggregation of newly translated proteins in an Escherichia coli strain deficient of the chaperonin GroEL.

In a newly isolated temperature-sensitive lethal Escherichia coli mutant affecting the chaperonin GroEL, we observed wholesale aggregation of newly translated proteins. After temperature shift, transcription, translation, and growth slowed over two to three generations, accompanied by filamentation and accretion (in approximately 2% of cells) of paracrystalline arrays containing mutant chaperonin complex. A biochemically isolated inclusion body fraction contained the collective of abundant proteins of the bacterial cytoplasm as determined by SDS/PAGE and proteolysis/MS analyses. Pulse-chase experiments revealed that newly made proteins, but not preexistent ones, were recruited to this insoluble fraction. Although aggregation of "stringent" GroEL/GroES-dependent substrates may secondarily produce an "avalanche" of aggregation, the observations raise the possibility, supported by in vitro refolding experiments, that the widespread aggregation reflects that GroEL function supports the proper folding of a majority of newly translated polypeptides, not just the limited number indicated by interaction studies and in vitro experiments.

Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes.

The double-ring chaperonin GroEL and its lid-like cochaperonin GroES form asymmetric complexes that, in the ATP-bound state, mediate productive folding in a hydrophilic, GroES-encapsulated chamber, the so-called cis cavity. Upon ATP hydrolysis within the cis ring, the asymmetric complex becomes able to accept non-native polypeptides and ATP in the open, trans ring. Here we have examined the structural basis for this allosteric switch in activity by cryo-EM and single-particle image processing. ATP hydrolysis does not change the conformation of the cis ring, but its effects are transmitted through an inter-ring contact and cause domain rotations in the mobile trans ring. These rigid-body movements in the trans ring lead to disruption of its intra-ring contacts, expansion of the entire ring and opening of both the nucleotide pocket and the substrate-binding domains, admitting ATP and new substrate protein.

Loops in the central channel of ClpA chaperone mediate protein binding, unfolding, and translocation.

The cylindrical Hsp100 chaperone ClpA mediates ATP-dependent unfolding of substrate proteins bearing "tag" sequences, such as the 11-residue ssrA sequence appended to proteins translationally stalled at ribosomes. Unfolding is coupled to translocation through a central channel into the associating protease, ClpP. To explore the topology and mechanism of ClpA action, we carried out chemical crosslinking and functional studies. Whereas a tag from RepA protein crosslinked proximally to the flexible N domains, the ssrA sequence in GFP-ssrA crosslinked distally in the channel to a segment of the distal ATPase domain (D2). Single substitutions placed in this D2 loop, and also in two apparently cooperating proximal (D1) loops, abolished binding of ssrA substrates and unfolded proteins lacking tags and blocked unfolding of GFP-RepA. Additionally, a substitution adjoining the D2 loop allowed binding of ssrA proteins but impaired their translocation. This loop, as in homologous nucleic-acid translocases, may bind substrates proximally and, coupled with ATP hydrolysis, translocate them distally, exerting mechanical force that mediates unfolding.

A mutant chaperonin with rearranged inter-ring electrostatic contacts and temperature-sensitive dissociation.

The chaperonin GroEL assists protein folding through ATP-dependent, cooperative movements that alternately create folding chambers in its two rings. The substitution E461K at the interface between these two rings causes temperature-sensitive, defective protein folding in Escherichia coli. To understand the molecular defect, we have examined the mutant chaperonin by cryo-EM. The normal out-of-register alignment of contacts between subunits of opposing wild-type rings is changed in E461K to an in-register one. This is associated with loss of cooperativity in ATP binding and hydrolysis. Consistent with the loss of negative cooperativity between rings, the cochaperonin GroES binds simultaneously to both E461K rings. These GroES-bound structures were unstable at higher temperature, dissociating into complexes of single E461K rings associated with GroES. Lacking the allosteric signal from the opposite ring, these complexes cannot release their GroES and become trapped, dead-end states.

Substrate polypeptide presents a load on the apical domains of the chaperonin GroEL.

A conundrum has arisen in the study of the structural states of the GroEL-GroES chaperonin machine: When either ATP or ADP is added along with GroES to GroEL, the same asymmetric complex, with one ring in a GroES-domed state, is observed by either x-ray crystallographic study or cryoelectron microscopy. Yet only ATP/GroES can trigger productive folding inside the GroES-encapsulated cis cavity by ejecting bound polypeptide from hydrophobic apical binding sites during attendant rigid body elevation and twisting of these domains. Here, we show that this difference occurs because polypeptide substrate in fact presents a load on the apical domains, and, although ATP can counter this load effectively, ADP cannot. We monitored apical domain movement in real time by fluorescence resonance energy transfer (FRET) between a fixed equatorial fluorophore and one attached to the mobile apical domain. In the absence of bound polypeptide, addition of either ATP/GroES or ADP/GroES to GroEL produced the same rapid rate and extent of decrease of FRET (t(1/2) < 1 sec), reflecting similarly rapid apical movement to the same end-state and explaining the results of the structural studies, which were all carried out in the absence of substrate polypeptide. But in the presence of bound malate dehydrogenase or rhodanese, whereas similar rapid and extensive FRET changes were observed with ATP/GroES, the rate of FRET change with ADP/GroES was slowed by >100-fold and the extent of change was reduced, indicating that the apical domains opened in a slow and partial fashion. These results indicate that the free energy of gamma-phosphate binding, measured earlier as 43 kcal per mol (1 cal = 4.184 J) of rings, is required for driving the forceful excursion or "power stroke" of the apical domains needed to trigger release of the polypeptide load into the central cavity.

Role of the gamma-phosphate of ATP in triggering protein folding by GroEL-GroES: function, structure and energetics.

Productive cis folding by the chaperonin GroEL is triggered by the binding of ATP but not ADP, along with cochaperonin GroES, to the same ring as non-native polypeptide, ejecting polypeptide into an encapsulated hydrophilic chamber. We examined the specific contribution of the gamma-phosphate of ATP to this activation process using complexes of ADP and aluminium or beryllium fluoride. These ATP analogues supported productive cis folding of the substrate protein, rhodanese, even when added to already-formed, folding-inactive cis ADP ternary complexes, essentially introducing the gamma-phosphate of ATP in an independent step. Aluminium fluoride was observed to stabilize the association of GroES with GroEL, with a substantial release of free energy (-46 kcal/mol). To understand the basis of such activation and stabilization, a crystal structure of GroEL-GroES-ADP.AlF3 was determined at 2.8 A. A trigonal AlF3 metal complex was observed in the gamma-phosphate position of the nucleotide pocket of the cis ring. Surprisingly, when this structure was compared with that of the previously determined GroEL-GroES-ADP complex, no other differences were observed. We discuss the likely basis of the ability of gamma-phosphate binding to convert preformed GroEL-GroES-ADP-polypeptide complexes into the folding-active state.

Folding with and without encapsulation by cis- and trans-only GroEL-GroES complexes.

Although a cis mechanism of GroEL-mediated protein folding, occurring inside a hydrophilic chamber encapsulated by the co-chaperonin GroES, has been well documented, recently the GroEL-GroES-mediated folding of aconitase, a large protein (82 kDa) that could not be encapsulated, was described. This process required GroES binding to the ring opposite the polypeptide (trans) to drive release and productive folding. Here, we have evaluated this mechanism further using trans-only complexes in which GroES is closely tethered to one of the two GroEL rings, blocking polypeptide binding by that ring. In vitro, trans-only folded aconitase with kinetics identical to GroEL-GroES. Surprisingly, trans-only also folded smaller GroEL-GroES-dependent substrates, Rubisco and malate dehydrogenase, but at rates slower than the cis reaction. Remarkably, in vivo, a plasmid encoding a trans-only complex rescued a GroEL-deficient strain, but the colony size was approximately one-tenth that produced by wild-type GroEL-GroES. We conclude that a trans mechanism, involving rounds of binding to an open ring and direct release into the bulk solution, can be generally productive although, where size permits, cis encapsulation supports more efficient folding.