Peptidase Activity of the Escherichia coli Hsp31 Chaperone

Abstract

Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative Cys 184 , His 185 , and Asp 213 catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity. A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity toward bovine serum albumin and a peptidase activity toward two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear. In this study, we report that Hsp31 does not diplay any significant proteolytic activity but has peptidolytic activity. The aminopeptidase cleavage preference of Hsp31 is Ala > Lys > Arg > His, suggesting that Hsp31 is an aminopeptidase of broad specificity. Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase. The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, in concordance with the importance of the putative His 85 , His 122 , and Glu 90 metal-binding site revealed by crystallographic studies. An Hsp31-deficient mutant accumulates more 8 -12-mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro. Proteins interacting with Hsp31 have been identified by reverse purification of a crude E. coli extract on an Hsp31-affinity column, followed by SDSpolyacrylamide electrophoresis and mass spectrometry. The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu, and tryptophanase were all found to interact with Hsp31, thus substantiating the role of Hsp31 as both chaperone and peptidase.Every organism responds to a sudden increase in the environmental temperature by the overexpression of a set of highly conserved heat shock proteins (1, 2). Most of these heat shock proteins function either as molecular chaperones, assisting in protein folding and renaturation or as proteases which degrade proteins that are beyond rescue.Hsp31, 1 the hchA gene product (formerly known as YedU), is a heat-inducible homodimeric protein of 31-kDa subunits, which was recently shown to exhibit molecular chaperone activity (3, 4). It promotes the functional folding of citrate synthase, ␣-glucosidase, and alcohol dehydrogenase. It also prevents the aggregation at 43°C of citrate synthase and alcohol dehydrogenase and interacts specifically with unfolded proteins (3, 4). Although Hsp31 does not exhibit any ATPase activity, some of its chaperone activities are partially inhibited by ATP (3, 4). The crystal structure of Hsp31 was solved at 1.6 Å resolution and revealed a system of hydrophobic patches, canyons, and grooves, which may stabilize partially unfolded protein substrates and expla...

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

“…Future studies will determine if chaperone activity is an evolutionarily conserved function displayed in LAPs from other kingdoms. Because two microbial enzymes, with structures distinct from LAPs, have dual aminopeptidase and chaperone activities (25,31), it is intriguing to speculate that aminopeptidase and chaperone activities co-evolved. If confirmed, LAPs will add to the growing diversity of multifunctional aminopeptidases.…”

“…However, the tomato LAP-A has several compelling biochemical characteristics that are shared with molecular chaperones, including its stability, high temperature optimum (60 -70°C), high pH optimum (9.0), and induction by a wide range of stresses (16,30). Finally, two microbial proteins are known to possess both chaperone and aminopeptidase activity in vitro: the E. coli Hsp31 chaperone and a Shizosaccharomyes pombe aspartyl aminopeptidase (25,31).…”

“…Molecular chaperone activity has also been revealed in proteins with other primary biological functions, including thioredoxin, peroxidase, elongation factors (eEF1a and EF-TU), and several protein-degrading enzymes (23)(24)(25)(26)(27). For example, several plastid ATP-dependent proteases are molecular chaperones (28,29).…”

Fermentation and alternative respiration compensate for NADH dehydrogenase deficiency in a prokaryotic model of DJ-1-associated Parkinsonism

“…YajL belongs to the PfpI/Hsp31/DJ-1 superfamily which includes chaperones (Quigley et al, 2003;Sastry et al, 2002), peptidases (Malki et al, 2005) and the Parkinson's disease-associated protein DJ-1 (Canet-Avilés et al, 2004;Cookson, 2005). The crystal structures of YajL and DJ-1 are strikingly similar (Wilson et al, 2003(Wilson et al, , 2005, and their backbone structures are essentially identical (0.9 Å C a root-mean-square deviation), suggesting that they have similar functions.…”

Reverse Structural Genomics

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