Substrate binding site flexibility of the small heat shock protein molecular chaperones

Abstract

Small heat shock proteins (sHSPs) serve as a first line of defense against stress-induced cell damage by binding and maintaining denaturing proteins in a folding-competent state. In contrast to the well-defined substrate binding regions of ATP-dependent chaperones, interactions between sHSPs and substrates are poorly understood. Defining substrate-binding sites of sHSPs is key to understanding their cellular functions and to harnessing their aggregation-prevention properties for controlling damage due to stress and disease. We incorporated a photoactivatable cross-linker at 32 positions throughout a well-characterized sHSP, dodecameric PsHsp18.1 from pea, and identified direct interaction sites between sHSPs and substrates. Model substrates firefly luciferase and malate dehydrogenase form strong contacts with multiple residues in the sHSP N-terminal arm, demonstrating the importance of this flexible and evolutionary variable region in substrate binding. Within the conserved ␣-crystallin domain both substrates also bind the ␤-strand (␤7) where mutations in human homologs result in inherited disease. Notably, these binding sites are poorly accessible in the sHSP atomic structure, consistent with major structural rearrangements being required for substrate binding. Detectable differences in the pattern of cross-linking intensity of the two substrates and the fact that substrates make contacts throughout the sHSP indicate that there is not a discrete substrate binding surface. Our results support a model in which the intrinsicallydisordered N-terminal arm can present diverse geometries of interaction sites, which is likely critical for the ability of sHSPs to protect efficiently many different substrates.alpha-crystallin ͉ cross-linking ͉ intrinsic disorder ͉ P-benzoylphenylalanine ͉ protein-protein interactions

Conserved, Disordered C Terminus of DnaK Enhances Cellular Survival upon Stress and DnaK in Vitro Chaperone Activity

“…This model is consistent with the finding that IDRs frequently act as flexible tethers and present molecular recognition features; the resulting flexibility enables the same regions to engage in multiple modes of binding (51), as suggested here. For example, the N-terminal disordered region of the small heat shock protein PsHsp18.1 allows the molecule to recognize a variety of misfolded substrates (52), as do the N-and C-terminal disordered regions of the ubiquitin ligase San1 (53).…”

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

“…This part of Hsp21 is a disordered, methionine-rich domain and has previously been suggested to be substratebinding (Harndahl et al 2001). For other sHsps, the Nterminal region has also previously been shown to be implicated in substrate-binding (Jaya et al 2009;Sharma et al 2000). Residues M1 and K27, and K18, are the only aminegroup-containing amino acid residues out of the 81 amino acid residues in the disordered N-terminal region of wild- Fig.…”

The functional roles of the unstructured N- and C-terminal regions in αB-crystallin and other mammalian small heat-shock proteins

“…Similarly, the N-terminal region of Hsp20 (HspB6) has multiple sites of interaction with a target protein as well as a role in regulating chaperone activity ) whilst also being important in the formation of a hetero-oligomer with Hsp27 (Heirbaut et al 2016). There is evidence from interactome studies that the Nterminal region of plant sHsps is involved in interacting with amorphously aggregating target proteins (Jaya et al 2009). Another example of the unstructured Nterminal region of sHsps' involvement in interacting with other proteins comes from the recent work of Sluchanko et al (2017) who determined the crystal structure of a complex between a phosphorylated form (at Ser16) of the dimeric sHsp, Hsp20 (van de Klundert et al 1998;Weeks et al 2014) and the 14-3-3σ dimer, i.e.…”