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 alpha-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 alpha-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 alpha-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.
Highlights
As the ␣-crystallin domain [1]
The most well documented aspect of sHSP-substrate interaction comes from multiple experiments showing that sHSP substrate binding capacity is enhanced by structural changes that expose hydrophobic surfaces that are normally occluded in the native sHSP oligomeric structure [1]
Experiments concluding that this domain interacts with substrates have included cross-linking to substrate [19, 20], identifying peptides that bind the hydrophobic probe (1,1Ј-bi(4-anilino)naphthalene5,5-disulfonic acid) (Bis-ANS) (20 –22), single amino acid substitutions [23,24,25,26], substrate binding to ␣-crystallin peptide arrays [27], and ability of peptides corresponding to a region of the ␣-crystallin domain to effect substrate protection [20, 27, 28]
Summary
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 [1,2,3]. SHSPs have been reported to act as negative regulators of apoptosis, to modulate cellular redox state, and to be linked to increased organism longevity [8,9,10] Consistent with their proposed chaperone function, sHSPs are associated with protein aggregates in a number of human diseases, including cataract, neurogenerative diseases, and myopathies [11]. There is good evidence that the ␣-crystallin domain shares a common structure in all members of this family consisting of a seven-stranded, IgG-like -sandwich with topology identical to p23 [1, 17, 18] Experiments concluding that this domain interacts with substrates have included cross-linking to substrate [19, 20], identifying peptides that bind the hydrophobic probe (1,1Ј-bi(4-anilino)naphthalene5,5-disulfonic acid) (Bis-ANS) (20 –22), single amino acid substitutions [23,24,25,26], substrate binding to ␣-crystallin peptide arrays [27], and ability of peptides corresponding to a region of the ␣-crystallin domain to effect substrate protection [20, 27, 28]. The N-terminal arms would be free to bind substrates when the sHSP oligomer dissociates [18]
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