The built-in lid couples ATP hydrolysis to substrate folding in archaeal group II chaperonins. (a) ATP hydrolysis but not ATP binding induces lid closure in M. maripaludis Cpn. Lid closure in the presence of ATP was monitored for Cpn WT, the ATPase-deficient mutant Cpn D386A and Cpn Δlid, as in Figure 1b⁹. (b) Homology model of a single subunit of Cpn WT and Cpn Δlid, respectively. The equatorial domain (Equ, black) is linked to the apical domain (Ap, light gray) via the flexible intermediate domain (Int, gray). Position of Asp386 is indicated. In Cpn Δlid, a short linker replaces the apical protrusions (lid, black). (c) ATP hydrolysis by Cpn WT, Cpn Δlid and Cpn D386A measured at 0.5 mM [α-³²P]ATP. (d) Cpn Δlid is able to bind denatured [³⁵S]rhodanese ([³⁵S]rhod) but cannot efficiently promote rhodanese folding in the presence of ATP. Error bars represent s.e.m. Inset shows an autoradiogram of [³⁵S]rhodanese–chaperonin complexes analyzed by 4% nondenaturing gel electrophoresis.

Negative allosteric coupling between rings affects ATP binding and hydrolysis. (a,b) Negative allosteric coupling between rings results in a second allosteric transition occurring at high ATP concentrations in wild-type chaperonins. Initial velocities of ATP hydrolysis by TRiC (a) and Cpn WT (b) were plotted against ATP concentration and fit to equation (2) (see Methods). Each data point shows the average of at least three independent experiments. Error bars represent s.e.m. (c) Proposed conformational states for group II chaperonins at different ATP concentrations. T/T state: in the absence of nucleotide, both rings reside in the T state, and the lid structure is not formed. This T/T complex has a low affinity for ATP. R/T′ state: at intermediate ATP concentrations (0.2 mM ATP), the cis-ring reaches saturation for ATP binding and hydrolysis and therefore assumes the R state. The trans-ring adopts a conformational state T′ with low affinity for ATP as a consequence of negative cooperativity between the rings. This asymmetric state has optimal ATPase activity (v_max(1)). R′/R′ state: at high ATP concentrations (1 mM ATP), the negative cooperativity is overcome and both rings bind and hydrolyze ATP simultaneously. The two rings hinder each other, leading to the observed drop in the hydrolysis rate v_max.

The built-in lid affects inter-ring communication of group II chaperonins. (a) Nested allosteric coupling in group II chaperonins. Positive cooperativity among the subunits of one ring is nested into negative cooperativity between the two rings. (b,c) The second allosteric transition, occurring at high ATP concentrations, is absent in the lidless chaperonin variants. Initial velocities of ATP hydrolysis by cTRiC (b) and Cpn Δlid (c) were plotted against ATP concentration and fit to equation (2) (see Methods). Each data point shows the average of at least three independent experiments. Error bars represent s.e.m.

The built-in lid couples ATP hydrolysis to substrate folding in the eukaryotic chaperonin TRiC. (a) cTRiC is a lidless version of TRiC generated by a selective cleavage within the apical protrusions²⁷. Prot K, proteinase K. (b) SDS-PAGE analysis followed by Coomassie staining of TRiC and cTRiC. Cleavage in the apical protrusion of each of the eight different 55- to 60-kDa subunits of TRiC produces sixteen 24- to 36-kDa fragments²⁷. (c) cTRiC is able to bind denatured [³⁵S]actin but cannot promote [³⁵S]actin folding in the presence of ATP. About 60% of bound [³⁵S]actin was refolded by TRiC in the presence of ATP (lane 3). Lane 1, native [³⁵S]actin migration standard. (d) ATP hydrolysis by TRiC and cTRiC measured at 0.6 mM [α-³²P]ATP.

The built-in lid is required for positive cooperativity between the subunits of one ring. (a) Two possible models for allosteric coupling of subunits in group II chaperonins. In the group I chaperonin GroEL, intra-ring allosteric coupling is achieved by interactions between the apical and intermediate domains of neighboring subunits. In group II chaperonins, two models are possible. Model 1: intra-ring coupling is independent of the lid, similar to GroEL. Model 2: the lid segments are required to orchestrate synchronized action between subunits in one ring. (b,c) Wild-type eukaryotic and archaeal chaperonins show positive cooperativity among the subunits of one ring. Initial velocities of ATP hydrolysis by TRiC (b) and Cpn WT (c) were plotted against ATP concentration and fit to the Hill equation (equation (2), see Methods). Each data point shows the average of at least three independent experiments. Error bars represent s.e.m. (d) Model of the ATP-induced conformational change of one ring of TRiC and Cpn WT. Positive cooperativity causes subunits in one ring to undergo a concerted conformational change leading to lid closure. (e,f) Allosteric coupling of subunits in one ring is impaired in a lidless chaperonin. Initial velocities of ATP hydrolysis by cTRiC (e) and Cpn Δlid (f) were plotted against ATP concentration and fit to equation (2) as above. Each data point shows the average of at least three independent experiments. Error bars represent s.e.m. (g) Model of the ATP-induced conformational changes in lidless cTRiC and Cpn Δlid. No allosteric coupling between the subunits can be observed, indicating that the subunits bind ATP independently of one another.

Group II chaperonins sample two different conformational states at intermediate and high ATP concentrations. (a) In the absence of nucleotide, chaperonins predominantly sample the T/T state, in which both rings are in the open conformation and therefore susceptible to protease digestion. TRiC and Cpn WT are fully protected from protease cleavage at 1 mM ATP-AlF_x and therefore reside in a symmetrically closed complex, the R′/R′ state. The partial protection pattern at 0.2 mM ATP-AlF_x indicates the existence of an asymmetrically closed R/T′ state at intermediate ATP concentrations. (b,c) The asymmetric R/T′– state of group II chaperonins supports optimal substrate folding. b shows TRiC-mediated [³⁵S]actin folding at 1 and 0.2 mM ATP, examined by protease digestion (left) and nondenaturing PAGE (right), followed by autoradiography. FL, full-length. c shows substrate-folding rates of TRiC and Cpn at 0.2 and 1 mM ATP. Left, time course of actin folding by TRiC in the absence of nucleotide (5 mM EDTA) and in the presence of 0.2 and 1 mM ATP. Right, time course of rhodanese folding by Cpn WT in the absence of nucleotide (5 mM EDTA) and in the presence of 0.2 and 1 mM ATP. Error bars represent s.e.m.

The built-in lid controls the ATPase cycle of group II chaperonins. ATP binding to subunits in one ring is a cooperative event communicated by the apical protrusions. Subsequent ATP hydrolysis within the cis-ring is accompanied by a synchronized conformational change that results in lid closure⁹. The lid probably remains closed in the post-hydrolysis ADP + P_i state. Reopening of the stable iris-like lid structure seems to be the rate-limiting step in the conformational cycle¹⁸. Owing to negative inter-ring cooperativity^14,17,18, ATP hydrolysis in the trans-ring can occur only once the products of ATP hydrolysis have dissociated from the cis-ring. The precise mechanism that unravels the stable lid structure remains undefined.