How GroES regulates binding of nonnative protein to GroEL

At present, it is still enigmatic how the reaction cycle by which the Escherichia coli GroE chaperones mediate protein folding in the cell is coordinated with respect to the sequential order of binding and release of GroES, nucleotide, and nonnative protein. It is generally assumed that the asymmetric GroEL-GroES complex is the acceptor state for substrate protein. Nevertheless, this species is poorly understood in its binding characteristics for nucleotide and nonnative protein. We show here that this species has a high affinity binding site for nonnative protein. In addition to this, binding of nucleotide to one GroEL ring is strongly favored by GroES binding to the other ring. However, the slow rate of release of substrate protein from the unproductive trans-position kinetically favors the binding of a second GroES, thereby forming a symmetric GroEL₁₄·(GroES₇)₂ complex and simultaneously ensuring that substrate protein is sequestered in a position underneath GroES. Our results demonstrate that the intrinsic binding characteristics of the trans-bullet complex determine the sequence of events during the reaction cycle.