30th Annual SSRL Users' Meeting — October 9-10, 2003

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.