Nested cooperativity in the ATPase activity of the oligomeric chaperonin GroEL

Abstract

Initial rates of ATP hydrolysis by wild-type GroEL were measured as a function of ATP concentration from 0 to 0.8 mM. Two allosteric transitions are observed: one at relatively low ATP concentrations (< or = 100 microM) and the second at higher concentrations of ATP with respective midpoints of about 16 and 160 microM. Two allosteric transitions were previously observed also in the case of the Arg-196-->Ala GroEL mutant [Yifrach, O., & Horovitz, A. (1994) J. Mol. Biol. 243, 397-401]. On the basis of these observations a mathematical model for nested cooperativity in ATP hydrolysis by GroEL is developed in which there are two levels of allostery: one within each ring and the second between rings. In the first level, each hepatameric ring is in equilibrium between the T and R states, in accordance with the Monod-Wyman-Changeux (MWC) model of cooperativity [Monod et al. (1965) J. Mol. Biol. 12, 88-118]. A second level of allostery is between the rings of the GroEL particle which undergoes sequential Koshland-Némethy-Filmer (KNF)-type transitions from the TT state via the TR state to the RR state [Koshland et al. (1966) Biochemistry 5, 365-385]. Using our model, we estimate the values of the Hill coefficient for the negative cooperativity between rings in wild-type GroEL and the Arg-196-->Ala mutant to be 0.003 (+/- 0.001) and 0.07 (+/- 0.02), respectively. The inter-ring coupling free energies in wild-type GroEL and the Arg-196-->Ala mutant are -7.5 (+/- 0.4) and -3.9 (+/- 0.3) kcal mol-1, respectively.

Requirement for binding multiple ATPs to convert a GroEL ring to the folding-active state

“…In particular, ATP drives the final step of information transfer for many genes, the step of chaperonin-mediated protein folding (1,2). The GroEL double ring machine, for example, is directed into its folding-active state by the concerted binding of ATPs to the 7 subunits of a substrate polypeptide-bound ring (3). ATP binding in the equatorial domains of these subunits drives rigid body movements of the apical domains, which bear the hydrophobic polypeptide-binding surface lining the opening to a ring (4,5).…”

Substrate polypeptide presents a load on the apical domains of the chaperonin GroEL

“…After hydrolysis, ligands, including polypeptide, in either folded or nonnative forms (15,16), are released from the ring by the binding of ATP to the opposite ring (14), a function of the anticooperativity of ATP binding between rings (17), which places them out of phase with respect to each other in actions of polypeptide binding and folding. Thus, at the chaperonin machine, as for others, the energy of ATP binding is used to effect work, here either of nucleating the folding-active state when binding to the same ring as polypeptide or of dissociating the folding-active state when binding to the opposite ring.…”

Dynamics, flexibility, and allostery in molecular chaperonins

“…The high cooperativity of ATP binding and hydrolysis in the GroEL/ES system, revealed by early functional and kinetic studies [22][23][24], was later associated to Table 1 Chaperonin classification (adapted from [8] structural changes [25]. Subsequent analyses described both positive (within a ring) and negative cooperativity (between rings) [17,20,26,27]. Interestingly, the positive intra-ring cooperativity has been shown to be present only for ATP.…”

“…The cycle is powered by ATP binding and hydrolysis, and is regulated by the complex interplay of positive and negative cooperativity [22,27]. The main steps of the reaction cycle have been established by extensive structural and functional studies over the last two decades.…”