Evidence for an essential function of the N terminus of a small heat shock protein in vivo , independent of in vitro chaperone activity

Abstract

To investigate the mechanism of small heat shock protein (sHsp) function, unbiased by current models of sHsp chaperone activity, we performed a screen for mutations of Synechocystis Hsp16.6 that reduced the ability of the protein to provide thermotolerance in vivo. Missense mutations at 17 positions throughout the protein and a C-terminal truncation of 5 aa were identified, representing the largest collection of sHsp mutants impaired in function in vivo. Ten mutant proteins were purified and tested for alterations in native oligomeric structure and in vitro chaperone activity. These biochemical assays separated the mutants into two groups. The C-terminal truncation and six mutations in the ␣-crystallin domain destabilized the sHsp oligomer and reduced in vitro chaperone activity. In contrast, the other three mutations had little effect on oligomer stability or chaperone activity in vitro. These mutations were clustered in the N terminus of Hsp16.6, pointing to a previously unrecognized, important function for this evolutionarily variable domain. Furthermore, the fact that the N-terminal mutations were impaired in function in vivo, but active as chaperones in vitro, indicates that current biochemical assays do not adequately measure essential features of the sHsp mechanism of action.crystallin ͉ dimer ͉ Synechocystis ͉ thermotolerance ͉ heat stress

Plant Leucine Aminopeptidases Moonlight as Molecular Chaperones to Alleviate Stress-induced Damage

“…S7). The E429R protein protected NdeI activity at 0.4 M, whereas the K354E and D357R proteins protected NdeI at concentrations as low as 0.2 M. These data were in marked contrast with oligomeric structure mutants of HSP16.6 from Synechocystis, where the oligomeric stability of HSP16.6 is required for chaperone activity in vitro (43,44).…”

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.…”

“…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.…”

Small heat-shock proteins: important players in regulating cellular proteostasis

“…Various studies have suggested that target protein binding is mediated by the N-terminal domain [95][96][97][98] or the ACD [99][100][101]. Indeed, with regards to the latter, we [18] and others [17,20,102] have shown that isolated ACDs exhibit chaperone function.…”