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

Abstract

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 ␣-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 sHSPsubstrate 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 ␣-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 ␣-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.The small heat shock proteins (sHSPs), 4 and the related ␣-crystallins comprise a superfamily of chaperones defined by a conserved C-terminal domain of ϳ90 amino acids referred to as the ␣-crystallin domain (1). Flanking this domain is a short C-terminal extension and an N-terminal arm of variable length and highly divergent sequence (1-3). Although the monomeric size of the sHSPs ranges from 15 to 42 kDa, in their native state most sHSPs assemble into large oligomers of 8 to Ͼ32 subunits, although there are also dimeric and tetrameric sHSPs (1, 4). 5Studies with numerous sHSPs from different organisms have shown that in vitro these proteins act as chaperones by binding to partially unfolded proteins in an ATP-independent manner, preventing their irreversible aggregation (1, 5). Substrates that are denatured in the presence of sHSPs can then be refolded and reactivated by the ATP-dependent chaperone DnaK/ Hsp70 with the participation in some cases of ClpB/Hsp100 and GroEL (1, 6, 7). In vivo, increased expression of these ubiquitous stress proteins can enhance cellular tolerance to a variety of stresses, such as heat, salt, drugs, and oxidants (1). sHSPs have also been reported to act as negative regulators of apoptosis, to modulate cellular redox state, and to be linked to increased orga...

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

“…3 and 4). Although an essential function for the sHSP N-terminal arm in substrate protection has been proposed (2,(20)(21)(22)(23)28), our data provide direct evidence that the N-terminal arm binds substrate. We also show that regions of the ␣-crystallin domain and C-terminal extension form substrate specific crosslinks, but at a lower intensity compared with those of the N-terminal arm (Fig.…”

“…While MDH formed cross-links with this region, Luc showed little or no interaction. Differential interaction of MDH and Luc with the ␣-crystallin domain was also observed in experiments using chimeric sHSPs in which identity of the ␣-crystallin domain affected protection of MDH, but not of Luc (20). Even for MDH, however, the intensity of cross-linked species was low in this region, despite the fact that introducing Bpa at these sites would increase the surface hydrophobicity of the sHSP and thereby increase potential for interactions with hydrophobic substrate surfaces.…”

“…Chaperone activity of the Bpa variants was measured by their ability to protect the model substrates MDH and Luc from heat-induced aggregation. For MDH, wild-type or Bpasubstituted PsHsp18.1 was mixed with MDH at 1:1, 2:1, or 4:1 molar ratios (sHSP:MDH) and incubated for 120 min at 45°C, conditions which lead to full aggregation of MDH in the absence of sHSP (20). Luc protection was tested at molar ratios of 3:1, 6:1, or 12:1 (sHSP:Luc) and incubated for 8.5 min at 42°C.…”

“…Luc protection was tested at molar ratios of 3:1, 6:1, or 12:1 (sHSP:Luc) and incubated for 8.5 min at 42°C. Luc is more heat-sensitive than MDH and aggregates completely under these conditions in the absence of sHSP (20). After incubation, soluble and pellet fractions were analyzed by SDS/ PAGE.…”

“…Sequence variability and structural disorder, along with experimental evidence make the N-terminal arm a good candidate for substrate binding. Chaperone activity is altered in N-terminal chimeras, and in N-terminal point and deletion mutants, implicating the Nterminal arm in substrate protection (20)(21)(22)(23). However, these data do not distinguish between disruption of substrate interaction sites on the N-terminal arm, versus perturbation of some other sHSP property, such as oligomer integrity, which then indirectly impacts chaperone activity.…”