Eman Basha

Crystal structure and assembly of a eukaryotic small heat shock protein.

The 2.7 Å structure of wheat HSP16.9, a member of the small heat shock proteins (sHSPs), indicates how its α-crystallin domain and flanking extensions assemble into a dodecameric double disk. The folding of the monomer and assembly of the oligomer are mutually interdependent, involving strand exchange, helix swapping, loose knots and hinged extensions. In support of the chaperone mechanism, the substrate-bound dimers, in temperature-dependent equilibrium with higher assembly forms, have unfolded N-terminal arms and exposed conserved hydrophobic binding sites on the α-crystallin domain. The structure also provides a model by which members of the sHSP protein family bind unfolded substrates, which are involved in a variety of neurodegenerative diseases and cataract formation.

Small heat shock proteins and α-crystallins: dynamic proteins with flexible functions

The small heat shock proteins (sHSPs) and the related α-crystallins (αCs) are virtually ubiquitous proteins that are strongly induced by a variety of stresses, but that also function constitutively in multiple cell types in many organisms. Extensive research has demonstrated that a majority of sHSPs and αCs can act as ATP-independent molecular chaperones by binding denaturing proteins and thereby protecting cells from damage due to irreversible protein aggregation. As a result of their diverse evolutionary history, their connection to inherited human diseases, and their novel protein dynamics, sHSPs and αCs are of significant interest to many areas of biology and biochemistry. However, it is increasingly clear that no single model is sufficient to describe the structure, function or mechanism of action of sHSPs and αCs. In this review, we discuss recent data that provide insight into the variety of structures of these proteins, their dynamic behavior, how they recognize substrates, and their many possible cellular roles.

Quaternary dynamics and plasticity underlie small heat shock protein chaperone function

Small Heat Shock Proteins (sHSPs) are a diverse family of molecular chaperones that prevent protein aggregation by binding clients destabilized during cellular stress. Here we probe the architecture and dynamics of complexes formed between an oligomeric sHSP and client by employing unique mass spectrometry strategies. We observe over 300 different stoichiometries of interaction, demonstrating that an ensemble of structures underlies the protection these chaperones confer to unfolding clients. This astonishing heterogeneity not only makes the system quite distinct in behavior to ATP-dependent chaperones, but also renders it intractable by conventional structural biology approaches. We find that thermally regulated quaternary dynamics of the sHSP establish and maintain the plasticity of the system. This extends the paradigm that intrinsic dynamics are crucial to protein function to include equilibrium fluctuations in quaternary structure, and suggests they are integral to the sHSPs’ role in the cellular protein homeostasis network.

The Identity of Proteins Associated with a Small Heat Shock Protein during Heat Stress in Vivo Indicates That These Chaperones Protect a Wide Range of Cellular Functions

The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believed to prevent irreversible protein aggregation and to facilitate subsequent protein renaturation in cooperation with ATP-dependent chaperones. Although sHSP chaperone activity has been studied extensively in vitro, understanding the mechanism of sHSP function requires identification of proteins that are sHSP substrates in vivo. We have used both immunoprecipitation and affinity chromatography to recover 42 proteins that specifically interact with Synechocystis Hsp16.6 in vivo during heat treatment. These proteins can all be released from Hsp16.6 by the ATP-dependent activity of DnaK and co-chaperones and are heat-labile. Thirteen of the putative substrate proteins were identified by mass spectrometry and reveal the potential for sHSPs to protect cellular functions as diverse as transcription, translation, cell signaling, and secondary metabolism. One of the putative substrates, serine esterase, was purified and tested directly for interaction with purified Hsp16.6. Hsp16.6 effectively formed soluble complexes with serine esterase in a heat-dependent fashion, thereby preventing formation of insoluble serine esterase aggregates. These data offer critical insights into the characteristics of native sHSP substrates and extend and provide in vivo support for the chaperone model of sHSP function.

The N-terminal Arm of Small Heat Shock Proteins Is Important for Both Chaperone Activity and Substrate Specificity

Small heat shock proteins (sHSPs) are a ubiquitous class of molecular chaperones that interacts with substrates to prevent their irreversible insolubilization during denaturation. How sHSPs interact with substrates remains poorly defined. To investigate the role of the conserved C-terminal α-crystallin domain versus the variable N-terminal arm in substrate interactions, we compared two closely related dodecameric plant sHSPs, Hsp18.1 and Hsp16.9, and four chimeras of these two sHSPs, in which all or part of the N-terminal arm was switched. The efficiency of substrate protection and formation of sHSP-substrate complexes by these sHSPs with three different model substrates, firefly luciferase, citrate synthase, and malate dehydrogenase (MDH) provide new insights into sHSP/substrate interactions. Results indicate that different substrates have varying affinities for different domains of the sHSP. For luciferase and citrate synthase, the efficiency of substrate protection was determined by the identity of the N-terminal arm in the chimeric proteins. In contrast, for MDH, efficient protection clearly required interactions with the α-crystallin domain in addition to the N-terminal arm. Furthermore, we show that sHSP-substrate complexes with varying stability and composition can protect substrate equally, and substrate protection is not correlated with sHSP oligomeric stability for all substrates. Protection of MDH by the dimeric chimera composed of the Hsp16.9 N-terminal arm and Hsp18.1 α-crystallin domain supports the model that a dimeric form of the sHSP can bind and protect substrate. In total, results demonstrate that sHSP-substrate interactions are complex, likely involve multiple sites on the sHSP, and vary depending on substrate.

Mechanistic Differences between Two Conserved Classes of Small Heat Shock Proteins Found in the Plant Cytosol

The small heat shock proteins (sHSPs) and α-crystallins are highly effective, ATP-independent chaperones that can bind denaturing client proteins to prevent their irreversible aggregation. One model of sHSP function suggests that the oligomeric sHSPs are activated to the client-binding form by dissociation at elevated temperatures to dimers or other sub-oligomeric species. Here we examine this model in a comparison of the oligomeric structure and chaperone activity of two conserved classes of cytosolic sHSPs in plants, the class I (CI) and class II (CII) proteins. Like the CI sHSPs, recombinant CII sHSPs from three divergent plant species, pea, wheat, and Arabidopsis, are dodecamers as determined by nano-electrospray mass spectrometry. While at 35 to 45 °C, all three CI sHSPs reversibly dissociate to dimers, the CII sHSPs retain oligomeric structure at high temperature. The CII dodecamers are, however, dynamic and rapidly exchange subunits, but unlike CI sHSPs, the exchange unit appears larger than a dimer. Differences in dodecameric structure are also reflected in the fact that the CII proteins do not hetero-oligomerize with CI sHSPs. Binding of the hydrophobic probe bis-ANS and limited proteolysis demonstrate CII proteins undergo significant, reversible structural changes at high temperature. All three recombinant CII proteins more efficiently protect firefly luciferase from insolubilization during heating than do the CI proteins. The CI and CII proteins behave strictly additively in client protection. In total, the results demonstrate that different sHSPs can achieve effective protection of client proteins by varied mechanisms.

Real-time monitoring of protein complexes reveals their quaternary organization and dynamics.

The dynamics of protein complexes are crucial for their function yet are challenging to study. Here, we present a nanoelectrospray (nESI) mass spectrometry (MS) approach capable of simultaneously providing structural and dynamical information for protein complexes. We investigate the properties of two small heat shock proteins (sHSPs) and find that these proteins exist as dodecamers composed of dimeric building blocks. Moreover, we show that these proteins exchange dimers on the timescale of minutes, with the rate of exchange being strongly temperature dependent. Because these proteins are expressed in the same cellular compartment, we anticipate that this dynamical behavior is crucial to their function in vivo. Furthermore, we propose that the approach used here is applicable to a range of nonequilibrium systems and is capable of providing both structural and dynamical information necessary for functional genomics.

Insights into Small Heat Shock Protein and Substrate Structure during Chaperone Action Derived from Hydrogen/Deuterium Exchange and Mass Spectrometry

Small heat shock proteins (sHSPs) and the related α-crystallins are ubiquitous chaperones linked to neurodegenerative diseases, myopathies, and cataract. To better define their mechanism of chaperone action, we used hydrogen/deuterium exchange and mass spectrometry (HXMS) to monitor conformational changes during complex formation between the structurally defined sHSPs, pea PsHsp18.1, and wheat TaHsp16.9, and the heat-denatured model substrates malate dehydrogenase (MDH) and firefly luciferase. Remarkably, we found that even when complexed with substrate, the highly dynamic local structure of the sHSPs, especially in the N-terminal arm (>70% exchange in 5 s), remains unchanged. These results, coupled with sHSP-substrate complex stability, indicate that sHSPs do not adopt new secondary structure when binding substrate and suggest sHSPs are tethered to substrate at multiple sites that are locally dynamic, a feature that likely facilitates recognition and refolding of sHSP-bound substrate by the Hsp70/DnaK chaperone system. Both substrates were found to be stabilized in a partially unfolded state that is observed only in the presence of sHSP. Furthermore, peptide-level HXMS showed MDH was substantially protected in two core regions (residues 95–156 and 228–252), which overlap with the MDH structure protected in the GroEL-bound MDH refolding intermediate. Significantly, despite differences in the size and structure of TaHsp16.9-MDH and PsHsp18.1-MDH complexes, peptide-level HXMS patterns for MDH in both complexes are virtually identical, indicating that stabilized MDH thermal unfolding intermediates are not determined by the identity of the sHSP.

Dissecting Heterogeneous Molecular Chaperone Complexes Using a Mass Spectrum Deconvolution Approach

Small heat-shock proteins (sHSPs) are molecular chaperones that prevent irreversible aggregation through binding nonnative target proteins. Due to their heterogeneity, these sHSP:target complexes remain poorly understood. We present a nanoelectrospray mass spectrometry analysis algorithm for estimating the distribution of stoichiometries comprising a polydisperse ensemble of oligomers. We thus elucidate the organization of complexes formed between sHSPs and different target proteins. We find that binding is mass dependent, with the resultant complexes reflecting the native quaternary architecture of the target, indicating that protection happens early in the denaturation. Our data therefore explain the apparent paradox of how variable complex morphologies result from the generic mechanism of protection afforded by sHSPs. Our approach is applicable to a range of polydisperse proteins and provides a means for the automated and accurate interpretation of mass spectra derived from heterogeneous protein assemblies.

The Quaternary Organization and Dynamics of the Molecular Chaperone HSP26 Are Thermally Regulated

The function of ScHSP26 is thermally controlled: the heat shock that causes the destabilization of target proteins leads to its activation as a molecular chaperone. We investigate the structural and dynamical properties of ScHSP26 oligomers through a combination of multiangle light scattering, fluorescence spectroscopy, NMR spectroscopy, and mass spectrometry. We show that ScHSP26 exists as a heterogeneous oligomeric ensemble at room temperature. At heat-shock temperatures, two shifts in equilibria are observed: toward dissociation and to larger oligomers. We examine the quaternary dynamics of these oligomers by investigating the rate of exchange of subunits between them and find that this not only increases with temperature but proceeds via two separate processes. This is consistent with a conformational change of the oligomers at elevated temperatures which regulates the disassembly rates of this thermally activated protein.