Interactions between Small Heat Shock Protein Subunits and Substrate in Small Heat Shock Protein-Substrate Complexes

Abstract

Small heat shock proteins (sHSPs) are dynamic oligomeric proteins that bind unfolding proteins and protect them from irreversible aggregation. This binding results in the formation of sHSP-substrate complexes from which substrate can later be refolded. Interactions between sHSP and substrate in sHSP-substrate complexes and the mechanism by which substrate is transferred to ATP-dependent chaperones for refolding are poorly defined. We have established C-terminal affinity-tagged sHSPs from a eukaryote (pea HSP18.1) and a prokaryote (Synechocystis HSP16.6) as tools to investigate these issues. We demonstrate that sHSP subunit exchange for HSP18.1 and HSP16.6 is temperature-dependent and rapid at the optimal growth temperature for the organism of origin. Increasing the ratio of sHSP to substrate during substrate denaturation decreased sHSP-substrate complex size, and accordingly, addition of substrate to pre-formed sHSP-substrate complexes increased complex size. However, the size of pre-formed sHSP-substrate complexes could not be reduced by addition of more sHSP, and substrate could not be observed to transfer to added sHSP, although added sHSP subunits continued to exchange with subunits in sHSPsubstrate complexes. Thus, although some number of sHSP subunits within complexes remain dynamic and may be important for complex structure/solubility, association of substrate with the sHSP does not appear to be similarly dynamic. These observations are consistent with a model in which ATP-dependent chaperones associate directly with sHSP-bound substrate to initiate refolding. Small heat shock proteins (sHSPs)1 are ubiquitous stress proteins of 12-42 kDa that share a conserved C-terminal domain of ϳ100 amino acids (the ␣-crystallin domain) (1). The conserved ␣-crystallin domain is flanked by a variable length non-conserved N-terminal arm and a short C-terminal extension. In vivo and in vitro sHSP monomers assemble into native oligomers of 9 to Ͼ30 subunits, depending on the specific sHSP (2). sHSPs and the related vertebrate ␣-crystallins are proposed to function as molecular chaperones by preventing irreversible aggregation and insolubilization of denatured proteins (3-11). Many experiments have shown that sHSPs and ␣-crystallins have a large binding capacity for a variety of heat and chemically denatured model protein substrates (8,10,(12)(13)(14)(15). The models for sHSP function have been extended to suggest that substrates in sHSP-substrate complexes (here after referred to as "complexes") are held in a folding competent state, such that substrates can be reactivated by the ATP-dependent chaperone HSP70 (DnaK) and its respective co-chaperones (6, 9, 13, 14, 16).The x-ray structures of HSP16.5 from Methanococcus jannaschii, a 24-subunit oligomer (17), and HSP16.9 from Triticum aestivum (wheat), a 12-subunit oligomer (17, 18), reveal common features of sHSP structure. Despite limited sequence identity, these structures have a 1.5-Å root mean square difference between C-␣ atoms in the conserved ␣-crystallin domain, whic...

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

“…It has been demonstrated that at substrate denaturing temperatures the equilibrium between the oligomeric and dimeric form of PsHsp18.1 and a number of other sHSPs is shifted toward the dimer (2,10), suggesting this is the substrate-binding species. For other sHSPs increased temperatures result in more rapid subunit exchange, which would also facilitate binding to a suboligomeric species (35,36). Because of difficulties in observing the sHSPsubstrate interaction at high temperatures, substrate bound to a dimeric or other suboligomeric sHSP form has not been observed directly.…”

“…PsHsp18.1 single site mutants were generated using the Strategene quick change method (Stratagene) using a plasmid containing PsHsp18.1 with a C-terminal Strep tag (36). An amber stop codon was introduced at the desired position for Bpa incorporation and the mutant construct was transformed into BL21 E. coli cells along with the pSup-BpaRS-6TRN plasmid which was a generous gift from Dr. Peter Schultz (Scripps Institute, CA) (24).…”

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

“…Proteins with the Hsp18.1 N Terminus Protect Luc More Efficiently than Those with the Hsp16.9 N Terminus-As mentioned previously, we observed that Hsp18.1 is more effective than Hsp16.9 in protecting firefly Luc against heat insolubilization (14,15,21). To determine whether this difference could be due to differences in the N terminus, the wild-type and chimeric proteins were compared for their ability to protect Luc from insolubilization during heating.…”

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

“…This Hsp16.6-strep strain is fully isogenic to our wild-type strain, with the exception of the C-terminal tag on Hsp16.6. We have shown previously that the affinity tag does not significantly affect growth or viability under control or heat stress conditions compared with the wild-type strain (63). Wildtype Hsp16.6, ⌬hsp16.6, and Hsp16.6-strep cells were treated as described for the immunoprecipitation experiments (Fig.…”