The Chaperonin ATPase Cycle: Mechanism of Allosteric Switching and Movements of Substrate-Binding Domains in GroEL

Abstract

Chaperonin-assisted protein folding proceeds through cycles of ATP binding and hydrolysis by the large chaperonin GroEL, which undergoes major allosteric rearrangements. Interaction between the two back-to-back seven-membered rings of GroEL plays an important role in regulating binding and release of folding substrates and of the small chaperonin GroES. Using cryo-electron microscopy, we have obtained three-dimensional reconstructions to 30 A resolution for GroEL and GroEL-GroES complexes in the presence of ADP, ATP, and the nonhydrolyzable ATP analog, AMP-PNP. Nucleotide binding to the equatorial domains of GroEL causes large rotations of the apical domains, containing the GroES and substrate protein-binding sites. We propose a mechanism for allosteric switching and describe conformational changes that may be involved in critical steps of folding for substrates encapsulated by GroES.

GroEL‐Mediated protein folding

“…Inspection of GroEL in different conformational states both by electron microscopy (Roseman et al, 1996) and by comparison of X-ray structures of unliganded GroEL and a GroELGroES complex support the hypothesis that hinge-like motions The overall structure of the GroEL tetradecamer is shown in two views: from the top, looking into the central cavity; and from the side. The left panel gives the dimensions of the cylinder.…”

“…The suggestion that the GroES mobile loops at least in part bind to and compete for the same surface of GroEL as polypeptide is supported both by observations in electron microscopy of the site of contact (Chen et al, 1994;Roseman et al, 1996) and by mutational findings that the same channel-facing residues critical for polypeptide binding (Fig. 1B) are also critical for binding GroES .…”

“…Interestingly, the phosphate binding loop (87-91) is immediately adjacent in the primary sequence to the a-helix and adjoining loop that bear K105 and A109, which participate in the first contact. Recent electron microscopy studies have suggested that, in the presence of ATP, this contact becomes less resolvable, indicating that it may indeed be a line of movement linking ATP binding with distant conformational changes (Roseman et al, 1996).…”

“…Importantly, the binding of GroES to GroEL Acceptor State U or luc 9 TRANS leads to a doubling in volume of the central channel in the bound GroEL ring, as the result of opening of the GroEL apical domains to contact GroES ( Fig. 3; Chen et al, 1994;Roseman et al, 1996). Thus, non-native polypeptides of up to -60 kDa can be accommodated in this central space underneath GroES.…”

“…The elegant single-turnover experiments of Lorimer and coworkers (Todd et al, 1994) established that release of GroES requires the second ring, being driven by the binding and "quantized" hydrolysis of seven ATPs in this ring, which transmits a signal to the cis ring, allosterically, that leads to release. Recent electron microscopy studies (Roseman et al, 1996) suggest that such release may be promoted through further twisting of the apical domains, releasing contact of the remaining hydrophobic binding sites of GroEL from the mobile loops of GroES. Such quantized action highlights the notion that the rings of GroEL appear to function as single units in nucleotide binding and hydrolysis, reflecting strong positive cooperativity within a ring.…”