Toward a mechanism for GroELGroES chaperone activity : An ATPase-gated and-pulsed folding and annealing cage ( protein folding / hsp 60 / cpn 60 / barnase )

Free GroEL binds denatured proteins very tightly: it retards the folding ofbarnase 400-fold and catalyzes unfolding fluctuations in native barnase and its folding intermediate. GroEL undergoes an allosteric transition from its tight-binding T-state to a weaker binding R-state on the cooperative binding of nucleotides (ATP/ADP) and GroES. The preformed GroEL.GroES. nucleotide complex retards the folding of barnase by only a factor of 4, and the folding rate is much higher than the ATPase activity that releases GroES from the complex. Binding of GroES and nucleotides to a preformed GroEL.denatured-barnase complex forms an intermediately fast-folding complex. We propose the following mechanism for the molecular chaperone. Denatured proteins bind to the resting GroEL.GroES. nucleotide complex. Fastfolding proteins are ejected as native structures before ATP hydrolysis. Slow-folding proteins enter chaperoning cycles of annealing and folding after the initial ATP hydrolysis. This step causes transient release of GroES and formation of the GroEL denatured-protein complexes with higher annealing potential. The intermediately fast-folding complex is formed on subsequent rebinding of GroES. The ATPase activity of GroEL-GroES is thus the gatekeeper that selects for initial entry of slow-folding proteins to the chaperone action and then pumps successive transitions from the faster-folding R-states to the tighter-binding/stronger annealing T-states. The molecular chaperone acts as a combination offolding cage and an annealing machine. The molecular chaperone GroEL of Escherichia coli is essential for the folding of many proteins in vivo and in vitro (1, 2). It is a typical member of the cpn60 class of chaperonins and the hsp60 class of heat shock proteins, consisting of two stacked heptameric rings with a large central cavity (3, 4). The cochaperonin, GroES, a member of the heptameric cpnlO family, is an essential adjunct for the GroEL-mediated folding of some proteins in vitro (5). The functional form of the complex appears to be asymmetric, with one ring of seven GroES subunits at one end of GroEL (6-8), although this is controversial (9). The structural and biophysical properties of GroEL, its complex with nucleotides (ATP and ADP), and GroES have been probed by a number of highly ingenious experiments (for the most recent general review, see ref. 10, and for specific biophysical details, see ref. 7). But, its mechanism of action is still controversial because of insufficient of information about individual steps in the folding of substrates. Two different mechanistic roles have been proposed for the GroE complex. One is that it acts as a cage in which a newly synthesized protein can fold in isolation (11), thus avoiding problems of aggregating with other partly folded chains (1215). The other is that it functions as an ATP-driven unfolding machine that actively catalyses the unfolding of misfolded The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. intermediates (16) that are "kinetically trapped" and would otherwise fold too slowly or aggregate (17-20). Correctly folded structures would snap back and misfolded parts may refold correctly. It is also proposed for this mechanism that folding takes place after the release of proteins from GroEL (18, 19). The precise role of the ATPase activity in the chaperone function is not clear. Small proteins, whose mechanisms of folding in vitro have been well characterized, can be used to probe the details of individual steps in the folding in the presence of chaperones. Both the 110-residue ribonuclease barnase (21-23) and the 64-residue chymotrypsin inhibitor 2 (CI2) (24) fold while complexed with GroEL in vitro, consistent with the "folding cage" hypothesis. It has been suggested that barnase (and CI2) are models for the folding of domains in larger proteins (23). Experiments on barnase and CI2 have also provided evidence for an annealing function of GroEL. GroEL slows down the rate of folding of denatured CI2 because bonds between them are broken during the folding transition (24). Conversely, unfolding should be catalysed because those bonds are formed during the unfolding transition. There is direct evidence for barnase being transiently unfolded by GroEL from H/2Hexchange studies which show that GroEL catalyzes the exchange of deeply buried protons that require full unfolding for exchange to occur (25). We use the term "annealing" to mean the transient melting of (mis)folded structures, followed by their refolding. Annealing does not imply that the major GroEL-bound species is the fully unfolded species, although it could be so in some cases. Indeed, kinetic data indicate that the predominant state of denatured barnase that is bound to GroEL is a compact folding intermediate (22), so that the fully unfolded form that is detected by H/2H-exchange is a minor component. GroEL does not cause the observable unfolding of a compact folding intermediate in the folding pathway of an antibody fragment (26), but annealing could still be occurring in the complex. It is sufficient for the correction of misfolding that parts of the protein become only transiently fully unfolded. We have now investigated the effects of GroES and nucleotides and the order of addition of reagents on the rate of folding of barnase in the presence of GroEL. These results, together with published data from elsewhere, point toward a mechanism of action that explains the role of the ATPase activity and reconciles some of the conflicting proposals. MATERIALS AND METHODS The mutant W94Y (27) was used as the wild-type protein in this study because of its more convenient changes in fluorescence on folding and unfolding (23). GroEL and the mutants W94Y(S91A) and W94Y(D8A/D12A/R110A) of barnase were obtained as described (28). The concentration of W94Y Abbreviation: CI2, chymotrypsin inhibitor 2. *To whom reprint requests should be addressed.