Quaternary dynamics and plasticity underlie small heat shock protein chaperone function

Abstract

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.heterogeneity | mass spectrometry | polydispersity | protein dynamics | proteostasis S mall Heat Shock Proteins (sHSPs) are one of the least well understood classes of molecular chaperones, proteins which act to prevent or reverse improper protein associations (1). The importance of the sHSPs is evidenced by their almost ubiquitous expression (2), the presence of multiple sHSP genes in most organisms (3), and their dramatic up-regulation under stress conditions making them among the most abundant of cellular proteins (4). They are implicated in a range of disease states including cataract, cancer, myopathies, motor neuropathies, and neurodegeneration (5-8). The current view of their chaperone action is that they bind unfolding "client" proteins, thereby preventing their irreversible aggregation (9-12). These sHSP: client complexes then interact with ATP-dependent chaperones to allow refolding of the clients (9-12). Structural interrogation of the complexes they form with clients has however been hampered by their apparent heterogeneity, and their organization remains consequently very poorly defined (13-15).MS is an emergent technology for the structural biology of protein assemblies (16), allowing the interrogation of a wide range of biomolecular systems, including those complicated by polydispersity and dynamics (17, 18). Here we capitalize on these unique advantages to study the complexes formed between pea HSP18.1 and a model client protein, firefly luciferase (Luc). HSP18.1 represents the family of class I cytosolic plant sHSPs which accumulate at heat-shock temperatures (≥38°C) to ≈1% of the total cellular protein (19). Extensive in vitro studies have established that HSP18.1 is able to bind destabilized clients, enabling subsequent refolding by the HSP70 machinery (20,21). With the in vivo clients of HSP18.1 yet to be identified, Luc was chosen as it is extremely thermo-sensitive and has been used extensively in chaperone studies (12). Luc does not interact with HSP18.1 at room te...

Probing the transient interaction between the small heat-shock protein Hsp21 and a model substrate protein using crosslinking mass spectrometry

“…Further increasing possible complexity, it has been found that various human sHsps may form mixed heterooligomers (Mymrikov et al 2012). The stable protein complexes formed between sHsp and substrate proteins also appear to be polydisperse (Stengel et al 2010), with complexes of pea Hsp18.1 and substrate protein luciferase comprised of variable numbers of both sHsp and substrate protein, covering over 300 different stoichiometries.…”

Nature’s molecular sponges: Small heat shock proteins grow into their chaperone roles

“…Increasing temperature led to a shift to higher oligomers from 13 to 20 subunits each (6). The rate-limiting step in oligomer rearrangement is subunit exchange, which is enhanced at higher temperatures.…”

“…Once temperature is increased and subunit exchange is enhanced, client-molecule association with the sHSPs is favored, and oligomeric sHSP:client complexes are populated. Brief incubation of the sHSP with the model client-protein luciferase at 42°C led to a rapid disappearance of the free client pool accompanied by a shift to larger oligomeric sHSP/luciferase complexes (6). The kinetics of these processes point to a mechanism ( Fig.…”

Heterogeneity and dynamics in the assembly of the Heat Shock Protein 90 chaperone complexes

“…Particularly significant have been recent applications of nanoflow electrospray to polydisperse molecular chaperone-substrate complexes wherein a snapshot of all complexes present at a given time can be obtained within a single mass spectrum (26). This MS approach should therefore enable investigation of the dynamics of a protein system complicated by the formation of multiple species in solution.…”