Closing the Folding Chamber of the Eukaryotic Chaperonin Requires the Transition State of ATP Hydrolysis

A. S. Meyer,¹ J. R. Gillespie,¹ D. Walther,⁴ I. S. Millett,² S. Doniach,³ and J. Frydman¹

¹Department of Biological Sciences, Stanford University, Stanford, CA 94305
²Department of Chemistry, Stanford University, Stanford, CA 94305
³Department of Applied Physics, Stanford University, Stanford, CA 94305
⁴Incyte Genomics, Inc., 3160 Porter Dr., Palo Alto, CA 94304

The ATP-dependent chaperonin TRiC (TCP-1 ring complex) is proposed to mediate protein folding by opening and closing a "built-in" lid over its central cavity. Here we combine structural and biochemical analyses to elucidate the mechanism and function of lid closure. Nucleotide-free TRiC adopts an open conformation that binds unfolded substrates in its central cavity. We find that incubation with ATP indeed promotes lid formation, yielding a closed central cavity. Formation of this closed chamber is essential for substrate folding, as blocking lid closure does not impair ATP hydrolysis or substrate-binding but abolishes productive folding of the bound polypeptide. To define how the ATPase cycle drives lid closure, we employed nucleotide analogues that stabilize distinct pre- and post-hydrolysis states. ATP-analogues that mimic the pre-hydrolysis state do not generate the closed cavity and leave the bound substrate in an unstructured conformation. In contrast, lid closure is induced by a mimic of the trigonal-bipyramidal transition state of the hydrolysis reaction, thus confining the substrate in the central chamber. We conclude that closure of the "built-in" lid is triggered by the transition state of ATP hydrolysis and is essential for productive folding of substrate proteins.