Journal of Molecular Biology
Volume 375, Issue 4, 25 January 2008, Pages 1026-1039
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Structure and Orientation of T4 Lysozyme Bound to the Small Heat Shock Protein α-Crystallin

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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 α-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 α-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.

Introduction

Although an overall thermodynamic bias directs the folding of a nascent polypeptide chain to the native state, the progress along the free energy hypersurface can be impeded by frequent population of aggregation-prone intermediates.1, 2, 3, 4 Furthermore, these intermediates are continuously populated after folding is complete through equilibrium transitions from the native state.5 In long-lived cellular systems, such as the ocular lens, accumulation of temperature-sensitive mutations or posttranslational modifications enhances misfolding as well as the equilibrium population of nonnative intermediates. In the crowded cellular environment, aggregation of proteins with exposed hydrophobic surfaces is favored by the high concentration and the excluded volume effect.6, 7 The lens is a particularly extreme example with its minimal protein turnover and the consequent lack of repair of damaged, destabilized proteins.8, 9 The most common form of lens opacity, age-related nuclear cataract, is associated with the formation of protein aggregates.10

A universal cellular mechanism for coping with misfolded or nonnative proteins involves the expression of five classes of heat shock proteins that facilitate folding following the emergence of proteins from the ribosomes and subsequently monitor the level of aggregation-prone intermediates.4, 11 All molecular chaperones recognize and bind proteins with exposed hydrophobic surfaces, thereby suppressing their aggregation. The small heat shock protein (sHSP) superfamily consists of oligomeric proteins that have high binding capacity for nonnative proteins, reaching in some instances one-to-one binding of each subunit to a substrate of equal molecular weight.12 In the lens, two sHSPs, αA- and αB-crystallin, play a critical role in maintaining lens transparency. They form polydisperse oligomers that undergo subunit exchange.13, 14 By middle age, αA- and αB-crystallin disappear from the water-soluble fraction, signifying the exhaustion of chaperone capacity.15 In general, progressive shifts in the folding equilibria of proteins can overload the folding capacity of the cell and lead to protein aggregation.16

In the presence of folded but thermodynamically destabilized proteins, sHSPs act as stability “sensors” binding the most destabilized proteins at higher levels.17, 18 Recognition and binding of substrates by sHSPs require transition to an activated state,12, 17, 18 which for eukaryotic sHSPs involves the dissociation into small multimers.19, 20 The N-terminal domain has been implicated as the main substrate-binding region,21, 22 although contacts occur with the conserved α-crystallin domain.23 sHSPs bind destabilized mutants of T4 lysozyme (T4L) in two modes differing in affinity and capacity.18 The binding can be described by a thermodynamic model20 where the activated state of the sHSP binds nonnative T4L, thereby coupling the substrate unfolding equilibrium to sHSP activation. The model proposes that the two modes of binding correspond to the recognition of distinct conformations of T4L.18

A number of studies have explored the structure of proteins bound to α-crystallin in complexes formed under conditions that induce substrate aggregation. The substrate conformations were found to be primarily slowly aggregating molten globules and maintain significant levels of residual secondary structure.24, 25, 26, 27, 28 Because these assays are performed under nonequilibrium conditions, they may promote transient interactions with nonnative states leading to kinetically trapped substrates. Fast-aggregating substrate intermediates, on the other hand, may escape binding by the chaperone. Furthermore, most mutations or posttranslational modifications often lead to subtle increases in the free energy of unfolding (ΔGunf) in contrast to the extreme destabilization of the native state (i.e., a negative ΔGunf) characteristic of these assays. Thus, aggregation assays do not capture the predominant equilibrium interactions of chaperones within the cellular environment that must precede nucleation of aggregation.

Here, we report the first direct investigation of the structure and orientation of a substrate, T4L, bound to a sHSP, α-crystallin. Complexes of defined stoichiometry between α-crystallin and T4L are formed under conditions where T4L does not aggregate and the equilibrium population of its native state is orders of magnitude higher than that of the ensemble of unfolded states. Distance constraints between spectroscopic probes are used to compare the structures of native and α-crystallin-bound T4L. In addition, analysis of the local environment across the T4L sequence in the chaperone complex reveals preferential regions of interactions and the overall orientation. Together, the results provide a novel perspective on the mechanism of recognition and binding by sHSP.

Section snippets

Approach and general methodology

To determine the conformational state of T4L bound to α-crystallin, we analyzed proximities in pairs of spectroscopic probes introduced at selected sites that fingerprint the tertiary fold. The pairs consisted of either two nitroxide spin labels or a bimane and a Trp. Trp quenches bimane fluorescence in the distance range below 15 Å.29 Spin labels undergo dipolar coupling in the 5- to 20-Å range, which is manifested as broadening of the electron paramagnetic resonance (EPR) lineshape.30 Changes

Discussion

The spectroscopic analysis presented above captures the essential structural features of a model substrate bound to a sHSP. The fluorescence and EPR constraints reveal a loss of tertiary contacts defining the fold and relative orientation of both T4L domains. Specific segments of secondary structures are also disrupted making it unlikely that native-like conformations or even molten globule states are stably bound to the chaperone. Effectively, excess α-crystallin acts to unfold T4L-L99A, which

Materials

Monobromobimane was purchased from Molecular Probes. 3-(2-Iodoacetamide)-proxyl spin label was purchased from Sigma-Aldrich.

Cloning and site-directed mutagenesis

T4L mutants were created using complimentary oligonucleotide primers containing the desired mutation and amplified via polymerase chain reaction as previously described.32 All mutants were cloned into pET20b+ expression vector. Plasmid DNA sequencing from transformed cells confirmed the presence of the desired mutations and the absence of unwanted changes. In this paper,

Acknowledgements

This work was supported by the National Eye Institute, National Institute of Health grants R01-EY12683 to Hassane S. Mchaourab and T32-EY07135 to Derek P. Claxton. The authors thank Jared A. Godar for critical reading of the manuscript and assistance in Fig. 1, Fig. 7, Fig. 9, and Dr. Hanane A. Koteiche for assistance in mutagenesis, discussions and critical reading of the manuscript.

References (38)

  • H.A. Sathish et al.

    Mechanism of chaperone function in small heat-shock proteins. Fluorescence studies of the conformations of T4 lysozyme bound to alphaB-crystallin

    J. Biol. Chem.

    (2003)
  • K.C. Giese et al.

    Changes in oligomerization are essential for the chaperone activity of a small heat shock protein in vivo and in vitro

    J. Biol. Chem.

    (2002)
  • R. Shashidharamurthy et al.

    Mechanism of chaperone function in small heat shock proteins: dissociation of the HSP27 oligomer is required for recognition and binding of destabilized T4 lysozyme

    J. Biol. Chem.

    (2005)
  • J. Shi et al.

    Cryoelectron microscopy and EPR analysis of engineered symmetric and polydisperse Hsp16.5 assemblies reveals determinants of polydispersity and substrate binding

    J. Biol. Chem.

    (2006)
  • E. Basha et al.

    The N-terminal arm of small heat shock proteins is important for both chaperone activity and substrate specificity

    J. Biol. Chem.

    (2006)
  • K.K. Sharma et al.

    Functional elements in molecular chaperone a-crystallin: identification of binding sites in a B-crystallin

    Biochem. Biophys. Res. Commun.

    (1997)
  • J.A. Carver et al.

    The interaction of the molecular chaperone alpha-crystallin with unfolding alpha-lactalbumin: a structural and kinetic spectroscopic study

    J. Mol. Biol.

    (2002)
  • K. Rajaraman et al.

    The chaperone-like alpha-crystallin forms a complex only with the aggregation-prone molten globule state of alpha-lactalbumin

    Biochem. Biophys. Res. Commun.

    (1998)
  • T.M. Treweek et al.

    The small heat-shock chaperone protein, alpha-crystallin, does not recognize stable molten globule states of cytosolic proteins

    Biochim. Biophys. Acta

    (2000)
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