The archaeal ATPase complex PAN, the homolog of the eukaryotic 26S proteasome-regulatory ATPases, was shown to associate transiently with the 20S proteasome upon binding of ATP or ATPgammaS, but not ADP. By electron microscopy (EM), PAN appears as a two-ring structure, capping the 20S, and resembles two densities in the 19S complex. The N termini of the archaeal 20S alpha subunits were found to function as a gate that prevents entry of seven-residue peptides but allows entry of tetrapeptides. Upon association with the 20S particle, PAN stimulates gate opening. Although degradation of globular proteins requires ATP hydrolysis, the PAN-20S complex with ATPgammaS translocates and degrades unfolded and denatured proteins. Rabbit 26S proteasomes also degrade these unfolded proteins upon ATP binding, without hydrolysis. Thus, although unfolding requires energy from ATP hydrolysis, ATP binding alone supports ATPase-20S association, gate opening, and translocation of unfolded substrates into the proteasome, which can occur by facilitated diffusion through the ATPase.

In the eukaryotic 26S proteasome, the 20S particle is regulated by six AAA ATPase subunits, and in archaea by a homologous ring complex, PAN. To clarify the role of ATP in proteolysis, we studied how nucleotides bind to PAN. Although PAN has six identical subunits it binds ATPs in pairs, and its subunits exhibit three conformational states with high, low, or no affinity for ATP. When PAN binds two ATPγS molecules, or two ATPγS plus two ADP molecules it is maximally active in binding protein substrates, associating with the 20S particle, and promoting 20S gate-opening. However, binding of four ATPγS molecules reduces these functions. The 26S proteasome shows similar nucleotide dependence. These findings imply an ordered cyclical mechanism in which two ATPase subunits bind ATP simultaneously and dock into the 20S. These results can explain how these hexameric ATPases interact with and “wobble” on top of the heptameric 20S proteasome.

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Initial rates of ATP hydrolysis by the chaperonin containing TCP-1 (CCT) from bovine testis were measured as a function of ATP concentration. Two allosteric transitions are observed: one at relatively low concentrations of ATP (<100 M) and the second at higher concentrations of ATP. The data suggest that CCT has positive intra-ring cooperativity and negative inter-ring cooperativity in ATP hydrolysis, with respect to ATP, as previously observed in the case of GroEL. It is shown that the relatively weak positive intra-ring cooperativity found in the case of CCT may be due to heterogeneity in its subunit composition. Our results suggest that nested allosteric behavior may be common to chaperone double-ring systems.

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What are the mechanisms of ligand-induced allosteric transitions in proteins? A powerful method to characterize pathways and transition states of reactions is value analysis. A value is the ratio between the changes on a perturbation (e.g., mutation) in the activation and equilibrium free energies of a reaction. Here, value analysis is used to characterize the ATP-induced allosteric transitions of GroEL by using changes in ATP concentration as perturbations. GroEL consists of two stacked back-to-back heptameric rings that bind ATP with positive cooperativity within rings and negative cooperativity between rings. Evidence is presented for the existence of parallel pathways for the allosteric transition of each ring. In both allosteric transitions, there is an abrupt ATP-dependent switch from a pathway with ATP-binding sites in the transition state that are very similar to those of the initial T state ( ‫؍‬ 0) to a pathway with a value of Ϸ0.3. The value procedure outlined here should be useful in mapping the energy landscape of allosteric transitions of other proteins.nested allostery ͉ cooperativity ͉ chaperonins

A kinetic analysis of the ATP-dependent dissociation of wild-type GroEL and mutants from immobilized GroES was carried out using surface plasmon resonance. Excellent fits of the data were obtained using a double-exponential equation with a linear drift. Both the fast and slow observed dissociation rate constants are found to have a sigmoidal dependence on the concentration of ATP. The values of the Hill coefficients corresponding to the fast and slow observed rate constants of dissociation of wild-type GroEL and the Arg197-->Ala mutant are in good agreement with the respective values of the Hill coefficients previously determined for these proteins from plots of initial rates of ATP hydrolysis as a function of ATP concentration, in the presence of GroES. Our results are consistent with a kinetic mechanism for dissociation of the GroEL-GroES complex according to which GroES release takes place after an ATP-induced conformational change in the trans ring that is preceded by ATP hydrolysis and a subsequent conformational change in the cis ring. It is shown that the rate of complex dissociation increases with increasing positive cooperativity in ATP binding by the GroEL ring distal to GroES in the GroEL-GroES complex.

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