Revisiting the GroEL-GroES Reaction Cycle via the Symmetric Intermediate Implied by Novel Aspects of the GroEL(D398A) Mutant

Abstract

The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists in protein folding with the aid of GroES and ATP. It is believed that GroEL alternates the folding-active rings and that the substrate protein (and GroES) can bind to the open trans-ring only after ATP in the cis-ring is hydrolyzed. However, we found that a substrate protein prebound to the trans-ring remained bound during the first ATP cycle, and this substrate was assisted by GroEL-GroES when the second cycle began. Moreover, a slow ATP-hydrolyzing GroEL mutant (D398A) in the ATP-bound form bound a substrate protein and GroES to the trans-ring. The apparent discrepancy with the results from an earlier study (Rye, H. S., Roseman, A. M., Chen, S., Furtak, K., Fenton, W. A., Saibil, H. R., and Horwich, A. L. (1999) Cell 97, 325-338) can be explained by the previously unnoticed fact that the ATP-bound form of the D398A mutant exists as a symmetric 1:2 GroEL-GroES complex (the "football"-shaped complex) and that the substrate protein (and GroES) in the medium is incorporated into the complex only after the slow turnover. In light of these results, the current model of the GroELGroES reaction cycle via the asymmetric 1:1 GroEL-GroES complex deserves reexamination.

Repetitive Protein Unfolding by the trans Ring of the GroEL-GroES Chaperonin Complex Stimulates Folding

“…For example, recent work has suggested that non-native substrate protein can bind to the trans ring of an ATP bullet complex, an observation that conflicts with a central assumption of the standard reaction model (17). Other recent work has suggested that ADP release plays a central role in the reaction cycle (18,19), whereas still other studies have suggested that ATP binds to an open GroEL ring before non-native substrate protein (20).…”

Probing Open Conformation of GroEL Rings by Cross-linking Reveals Single and Double Open Ring Structures of GroEL in ADP and ATP

“…Sameshima et al (31) monitored the fluorescence resonance energy transfer between GroEL and GroES and found that nearly equivalent amounts of football and bullet complexes coexist in the mixture during the functional reaction cycle. Koike-Takeshita et al (32) reported that a slow ATPhydrolyzing GroEL mutant can bind substrate protein and GroES at both rings of the GroEL to generate a football complex. These two studies also suggested that the football complex is generated in ATP, and the bullet complex is in ADP.…”

Crystal structure of the human mitochondrial chaperonin symmetrical football complex

“…Notably, mHsp60 assembles into a double-ring structure that binds two mHsp10 molecules, one at each end of the tetradecamer, forming the symmetric chaperonin complex [mHsp60 14-(mHsp10 7 ) 2 complex], previously termed the "football complex." This complex was found to play an important role in the chaperonin protein-folding reaction cycle (34)(35)(36)(37)(38)(39)(40)(41). To distinguish between the two symmetric halves of the football, we called one of the halves the "north pole" and the other the "south pole" (the full nomenclature of subunits is provided in Fig.…”