Evidence that beta-tubulin induces a conformation change in the cytosolic chaperonin which stabilizes binding: Implications for the mechanism of action

The class II chaperonin CCT facilitates protein folding by a process that i s not well-understood. One striking feature of this chaperonin is its appar ent selectivity in vivo, folding only actin, tubulin, and several other pro teins. In contrast, the class I chaperonin GroEL is thought to facilitate t he folding of many proteins within Escherichia coli, It has been proposed t hat this apparent selectivity is associated with certain regions of a subst rate protein's primary structure. Using limiting amounts of beta-tubulin, b eta-tubulin mutants, and beta-tubulin/ftsZ chimeras, we assessed the contri bution of select regions of beta-tubulin to CCT binding. In a complementary study, we investigated inter-ring communication in CCT where we exploited polypeptide binding sensitivity to nucleotide to quantitate nucleotide bind ing. beta-Tubulin bound with a high apparent affinity to CCT in the absence of nucleotide (apparent K-D similar to 3 nM; its apparent binding free ene rgy, Delta G, ca. -11.8 kcal/mol). Despite this, the interactions appear to be weak and distributed throughout much of the sequence, although certain sites ("hot spots") may interact somewhat more strongly with CCT. Globally averaged over the beta-tubulin sequence, these interactions appear to contr ibute ca. -9 to -11 cal/mol per residue, and to account for no more than 50 -60% of the total binding free energy. We propose that a conformation chang e or deformation induced in CCT by substrate binding provides the missing f ree energy which stabilizes the binary complex. We suggest that by coupling CCT deformation with polypeptide binding, CCT avoids the need for high "in trinsic" affinities for its substrates. This strategy allows for dynamic in teractions between chaperonin and bound substrate, which may facilitate fol ding on the interior surface of CCT in the absence of nucleotide and/or pro ductive release of bound polypeptide into the central cavity upon subsequen t MgATP binding. CCT displayed negative inter-ring cooperativity like GroEL , When ring 1 of CCT bound MgATP or beta-tubulin, the affinity of ring 2 fo r polypeptide or nucleotide was apparently reduced similar to 100-fold.