Gly192 at hinge 2 site in the chaperonin GroEL plays a pivotal role in the dynamic apical domain movement that leads to GroES binding and efficient encapsulation of substrate proteins.

The subunit structure of chaperonin GroEL is divided into three domains; the apical domain, the intermediate domain, and the equatorial domain. Each domain has a specific role in the chaperonin mechanism. The 'hinge 2' site of GroEL contains three glycine residues, Gly192, Gly374, and Gly375, connecting the apical domain and the intermediate domain. In this study, to understand the importance of the hinge 2 amino acid residues in chaperonin function, we substituted each of these three glycine residues to tryptophan. The GroEL mutants G374W and G375W were functionally similar to wild-type GroEL. However, GroEL G192W showed a significant decrease in the ability to assist the refolding of stringent substrate proteins. Interestingly, from biochemical assays and characterization using surface plasmon resonance analysis, we found that GroEL G192W was capable of binding GroES even in the absence of ATP to form a very stable GroEL-GroES complex, which could not be dissociated even upon addition of ATP. Electron micrographs showed that GroEL G192W intrinsically formed an asymmetric double ring structure with one ring locked in the 'open' conformation, and it is postulated that GroES binds to this open ring in the absence of ATP. Trans-binding of both substrate protein and GroES was observed for this binary complex, but simultaneous binding of both substrate and GroES (a mechanism that ensures substrate encapsulation) was impaired. We postulate that alteration of Gly192 severely compromises an essential movement that allows efficient encapsulation of unfolded protein intermediates.