Abstract
Small heat shock proteins (sHSPs) are ubiquitous chaperones that bind and sequester non-native proteins preventing their aggregation. Despite extensive studies of sHSPs chaperone activity, the location of the bound substrate within the sHSP oligomer has not been determined. In this paper, we used cryoelectron microscopy (cryoEM) to visualize destabilized mutants of T4 lysozyme (T4L) bound to engineered variants of the small heat shock protein Hsp16.5. In contrast to wild type Hsp16.5, binding of T4L to these variants does not induce oligomer heterogeneity enabling cryoEM analysis of the complexes. CryoEM image reconstruction reveals the sequestration of T4L in the interior of the Hsp16.5 oligomer primarily interacting with the buried N-terminal domain but also tethered by contacts with the α-crystallin domain shell. Analysis of Hsp16.5-WT/T4L complexes uncovers oligomer expansion as a requirement for high affinity binding. In contrast, a low affinity mode of binding is found to involve T4L binding on the outer surface of the oligomer bridging the formation of large complexes of Hsp16.5. These mechanistic principles were validated by cryoEM analysis of an expanded variant of Hsp16.5 in complex with T4L and Hsp16.5-R107G, which is equivalent to a mutant of human αB-crystallin linked to cardiomyopathy. In both cases, high affinity binding is found to involve conformational changes in the N-terminal region consistent with a central role of this region in substrate recognition.
Highlights
Small heat shock proteins bind non-native proteins preventing their aggregation
Prior to cryoelectron microscopy (cryoEM) analysis, T4 lysozyme (T4L) complexes with Hsp16.5 variants were purified by size exclusion chromatography (SEC) to remove unbound T4L if any
Both SEC and cryoEM analyses indicate that Hsp16.5-WT can undergo expansion of the ␣-crystallin domain shell by incorporation of additional subunits, which leads to an increase in the volume of the interior cavity and presumably enables binding of more substrate molecules per oligomer
Summary
Small heat shock proteins (sHSPs) bind non-native proteins preventing their aggregation. A low affinity mode of binding is found to involve T4L binding on the outer surface of the oligomer bridging the formation of large complexes of Hsp16.5 These mechanistic principles were validated by cryoEM analysis of an expanded variant of Hsp16.5 in complex with T4L and Hsp16.5-R107G, which is equivalent to a mutant of human ␣B-crystallin linked to cardiomyopathy. In both cases, high affinity binding is found to involve conformational changes in the N-terminal region consistent with a central role of this region in substrate recognition. The significance of this work extends to the mechanism of disease-linked ␣A- and ␣B-crystallin mutants where increased affinity to T4L is accompanied by transition of the ensemble of oligomers to a larger size [38, 39]
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