Integrity of N‐ and C‐termini is important for E. coli Hsp31 chaperone activity

Abstract

Hsp31 is a stress-inducible molecular chaperone involved in the management of protein misfolding at high temperatures and in the development of acid resistance in starved E. coli. Each subunit of the Hsp31 homodimer consists of two structural domains connected by a flexible linker that sits atop a continuous tract of nonpolar residues adjacent to a hydrophobic bowl defined by the dimerization interface. Previously, we proposed that while the bowl serves as a binding site for partially folded species at physiological temperatures, chaperone function under heat shock conditions requires that folding intermediates further anneal to high-affinity binding sites that become uncovered upon thermally induced motion of the linker. In support of a mechanism requiring that client proteins first bind to the bowl, we show here that fusion of a 20-residue-long hexahistidine tag to the N-termini of Hsp31 abolishes chaperone activity at all temperatures by inducing reversible structural changes that interfere with substrate binding. We further demonstrate that extending the C-termini of Hsp31 with short His tags selectively suppresses chaperone function at high temperatures by interfering with linker movement. The structural and functional sensitivity of Hsp31 to lengthening is consistent with the high degree of conservation of class I Hsp31 orthologs and will serve as a cautionary tale on the implications of affinity tagging.

A Glutathione-independent Glyoxalase of the DJ-1 Superfamily Plays an Important Role in Managing Metabolically Generated Methylglyoxal in Candida albicans

“…Although all previously characterized Hsp31-like proteins are dimeric, E. coli Hsp31 contains a 45-amino acid N-terminal extension that results in a formation of a different dimer than observed for the yeast proteins YDR533C or YOR391C, which lack this extension (20,(37)(38)(39)(40)(41). This N-terminal 45-amino acid region has been shown to be important for chaperone activity of the E. coli Hsp31 (42,43), and its absence in YDR533C indicates a potential functional difference between these proteins. The structure of C. albicans Glx3 also lacks this 45-amino acid N-terminal extension but, unlike YDR533C and YOR391C, is a monomer in the crystal.…”

Biofabrication of ZnS:Mn luminescent nanocrystals using histidine, hexahistidine, and His-tagged proteins: A comparison study

“…To determine if a high local concentration of histidine residues would yield higher quality nanocrystals, we conducted similar experiments with MGSSHHHHHHSSGLVPA, a hexahistidine-containing peptide that is liberated upon thrombin cleavage of N-terminally His-tagged proteins cloned into plasmid pET28(+) [20]. Figure 1B shows that results were similar to those observed with the free amino acid except that luminescent material was produced at lower concentrations (100 μM to 10 mM).…”

“…Plasmids pNT-hchA and pCT-hchA which encode variants of the molecular chaperone Hsp31 with N- and C-terminal hexahistidine tags, respectively, have been described previously [20, 21]. …”

“…Wild type Hsp31, its hexahistidine-tagged variants and the MGSSHHHHHHSSGLVPA peptide were purified as before [20]. For His-TrxA purification, BL21(DE3) cells harboring pH-TrxA were grown at 37°C to mid-exponential phase in LB medium supplemented with 30 μg/ml kanamycin, transferred to 30°C for 10 min, and induced with 0.4 mM IPTG.…”

Cloning, expression, purification, crystallization and preliminary X-ray analysis of the 31 kDaVibrio choleraeheat-shock protein VcHsp31

“…All members of the DJ-1 superfamily are oligomers in nature, ranging from dimers to dodecamers (Fioravanti et al, 2008). EcHsp31, which is organized as a homodimer, is a weak amidopeptidase towards a large number of protein substrates (Sastry et al, 2009). EcHsp31 consists of 13 -strands, 12 -helices and various loops forming two -domains designated the A and P domains, while the putative catalytic triad consists of Cys-His-Asp residues.…”