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 is not well-understood. One striking feature of this chaperonin is its apparent selectivity in vivo, folding only actin, tubulin, and several other proteins. In contrast, the class I chaperonin GroEL is thought to facilitate the folding of many proteins within Escherichia coli. It has been proposed that this apparent selectivity is associated with certain regions of a substrate protein's primary structure. Using limiting amounts of beta-tubulin, beta-tubulin mutants, and beta-tubulin/ftsZ chimeras, we assessed the contribution 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 binding. beta-Tubulin bound with a high apparent affinity to CCT in the absence of nucleotide (apparent K(D) approximately 3 nM; its apparent binding free energy, DeltaG, 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 contribute 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 change or deformation induced in CCT by substrate binding provides the missing free energy which stabilizes the binary complex. We suggest that by coupling CCT deformation with polypeptide binding, CCT avoids the need for high "intrinsic" affinities for its substrates. This strategy allows for dynamic interactions between chaperonin and bound substrate, which may facilitate folding on the interior surface of CCT in the absence of nucleotide and/or productive release of bound polypeptide into the central cavity upon subsequent 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 for polypeptide or nucleotide was apparently reduced approximately 100-fold.
Dobrzynski
J K
JK
Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Sternlicht
M L
ML
Peng
I
I
Farr
G W
GW
Sternlicht
H
H
D000595:Amino Acid Sequence; D001665:Binding Sites; D018833:Chaperonins / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism
D014404:Tubulin; D018833:Chaperonins