Insights into the structure and functions of the alkaliphilic Bacillus lehensis g1 clpc protein in alkaline environment

Abstract

The heat shock protein 100 (Hsp100) ClpC is a member of the AAA+ protein superfamily that contributes to the maintenance of the cellular protein homeostasis by energy dependent proteolysis. The aim of this work was to elucidate the interaction of the alkaliphilic Hsp100 ClpC from Bacillus lehensis G1 (Bl-ClpC) and its adaptor protein MecA in order to postulate their functions. The ClpC N-terminal domain of B. lehensis (Bl-ClpCN) was crystallised and the structure was solved to 1.85 Å. The crystal structure of ClpCN was analysed and compared to that of the archetypal species Bacillus subtillis. Comparisons of the N-terminal ClpC-interacting and hexamerisation domains between both species showed that B. lehensis G1 had an insertion (of unknown function) of four amino acids at the loop between helix 4 and strand 2, which were located on the outer surface of the barrel-shaped molecule. Apart from being highly unique, this characteristic was conserved only in the newly-isolated alkaliphilic B. lehensis G1 and not in any of the previously-reported structures of N-terminal domains isolated from the Hsp100 family. Furthermore, information concerning the physicochemical basis of alkaliphilic ClpC is still unclear. Therefore, the biochemical properties of Bl-ClpC were characterised under varying pH, temperatures, salt concentrations, and metal ions. Bl- ClpC showed two features which were distinct from other proteins in the Hsp100 family which were high salt concentrations and mild acidic pH caused an increase in ATPase activity. Bl-ClpC activity was considered to be similar to that of a halophilic protein, which demonstrated increased activity in high concentrations of NaCl, which was a common characteristic of a highly acidic protein. pH-induced structural changes and Bl- ClpC stability have been investigated as well, and these changes correlated with ATPase activity modulations in different pH. There was little effect on the protein structure when the pH was lowered from 9 to 5. In alkaline pH (pH 9 - 11), the presence of an ɑ-helicaldominated molten globule state was reported. The function of the unique four-residue insertion at the ɑ4-β2 loop, which was absent in the B. subtilis ClpC orthologue, has been successfully elucidated by using the structure-guided mutation approach, whereby a deletion mutation devoid of residue 76-79 (Δ76-79) was constructed. Circular dichroism spectroscopy was used to evaluate the structural perturbations associated with the deletion. The results demonstrated that the precise configuration of the ɑ4-β2 loop was important for maintaining the structure and function of Bl-ClpC. Δ76-79 led to severe destabilisation as well as unfolding of the secondary structure of the protein, all of which decreased ATPase activity. The optimum temperature for Δ76-79 was 25°C instead of 45°C for Bl-ClpC. These findings showed that the additional four residues at the ɑ4-β2 loop were critical for Bl-ClpC‟s structure and function. Overall, Bl-ClpC exhibited distinct responses to salt stress and mild acidic pH, hence implying that environmental conditions and stress adaptations were important selective forces which gave rise to the divergence of Hsp100 ClpC from its alkaliphilic archetype.