Structure and Orientation of T4 Lysozyme Bound to the Small Heat Shock Protein α-Crystallin

Abstract

We have determined the structural changes that accompany the formation of a stable complex between a destabilized mutant of T4 lysozyme (T4L) and the small heat shock protein alpha-crystallin. Using pairs of fluorescence or spin label probes to fingerprint the T4L tertiary fold, we demonstrate that binding disrupts tertiary packing in the two domains as well as across the active-site cleft. Furthermore, increased distances between i and i+4 residues of helices support a model in which the bound structure is not native-like but significantly unfolded. In the confines of the oligomer, T4L has a preferential orientation with residues in the more hydrophobic C-terminal domain sequestered in a buried environment, while residues in the N-terminal domain are exposed to the aqueous solvent. Furthermore, electron paramagnetic resonance spectral line shapes of sites in the N-terminal domain are narrower than in the folded, unbound T4L reflecting an unstructured backbone and an asymmetric pattern of contacts between T4L and alpha-crystallin. The net orientation is not affected by the location of the destabilizing mutation consistent with the notion that binding is not triggered by recognition of localized unfolding. Together, the structural and thermodynamic data indicate that the stably bound conformation of T4L is unfolded and support a model in which the two modes of substrate binding originate from two discrete binding sites on the chaperone.

Small Heat Shock Protein Activity Is Regulated by Variable Oligomeric Substructure

“…A recent model of sHSP chaperone function suggests that sHSP oligomers exist in two forms with differential activity: a low affinity state and a high affinity state (3,20). We propose here that, in the case of the ␣-crystallins, these two forms correspond to oligomers with dimeric and monomeric substructure, respectively (Fig.…”

Cryoelectron Microscopy Analysis of Small Heat Shock Protein 16.5 (Hsp16.5) Complexes with T4 Lysozyme Reveals the Structural Basis of Multimode Binding

“…2A) of the Hsp16.5-WT/T4L sample show that this fraction consists of clustered Hsp16.5 particles. Previous studies suggested that low affinity bound T4L are likely to be on the outer shell of the oligomer exposed to solvent (29). These molecules can then mediate the clustering of Hsp16.5 particles.…”

“…Interpretation of these experiments is intrinsically difficult and hindered by the heterogeneity of the substrate proteins under the non-equilibrium conditions typical of aggregation-based assays. To overcome these limitations, Claxton et al (29) used a spectroscopic approach to characterize the structure of T4L bound to ␣-crystallin. They demonstrated that native tertiary contacts are disrupted and secondary structure elements are unfolded supporting the contention that the stably bound T4L conformation is extensively unfolded.…”

Crystallin proteins and amyloid fibrils

“…Recent studies by Mchaourab and colleagues on the interaction of destabilized T4 lysozyme mutants with sHsps have led to a proposed mechanism of sHsp chaperone action in which the equilibrium between the folding and unfolding of the target protein (T4 lysozyme) is coupled to the equilibrium between the dissociated (dimeric) and oligomeric states of α-crystallin [42][43][44]. α-Crystallin binds T4 lysozyme in two modes with high and low affinity.…”

“…α-Crystallin binds T4 lysozyme in two modes with high and low affinity. In this model, the dimeric form of the sHsp is the activated state which recognizes and binds to the destabilized target protein [44].…”