GroES in the asymmetric GroEL₁₄–GroES₇ complex exchanges via an associative mechanism

Abstract

The interaction of the chaperonin GroEL₁₄ with its cochaperonin GroES₇ is dynamic, involving stable, asymmetric 1:1 complexes (GroES₇⋅GroEL₇–GroEL₇) and transient, metastable symmetric 2:1 complexes [GroES₇⋅GroEL₇–GroEL₇⋅GroES₇]. The transient formation of a 2:1 complex permits exchange of free GroES₇ for GroES₇ bound in the stable 1:1 complex. Electrophoresis in the presence of ADP was used to resolve free GroEL₁₄ from the GroES₇–GroEL₁₄ complex. Titration of GroEL₁₄ with radiolabeled GroES₇ to molar ratios of 32:1 demonstrated a 1:1 limiting stoichiometry in a stable complex. No stable 2:1 complex was detected. Preincubation of the asymmetric GroES₇⋅GroEL₇–GroEL₇ complex with excess unlabeled GroES₇ in the presence of ADP demonstrated GroES₇ exchange. The rates of GroES₇ exchange were proportional to the concentration of unlabeled free GroES₇. This concentration dependence points to an associative mechanism in which exchange of GroES₇ occurs by way of a transient 2:1 complex and excludes a dissociative mechanism in which exchange occurs by way of free GroEL₁₄. Exchange of radiolabeled ADP from 1:1 complexes was much slower than the exchange of GroES₇. In agreement with recent structural studies, this indicates that conformational changes in GroEL₁₄ following the dissociation of GroES₇ must precede ADP release. These results explain how the GroEL₁₄ cavity can become reversibly accessible to proteins under in vivo conditions that favor 2:1 complexes.