Substrate binding and specificity appear as major forces in the functional diversification of eqolisins.

Abstract

BackgroundEqolisins are rare acid proteases found in archaea, bacteria and fungi. Certain fungi secrete acids as part of their lifestyle and interestingly these also have many eqolisin paralogs, up to nine paralogs have been recorded. This suggests functional redundancy and diversification, which was the subject of the research we performed and describe here.\n\nResultsWe identified eqolisin homologs by means of iterative HMMER analysis of the NR database. The identified sequences were scrutinized for which we defined novel hallmarks, identified by molecular dynamics simulations of mutants of highly conserved positions, using the structure of an eqolisin that was crystallized in the presence of a transition state inhibitor. Four conserved glycines were shown to be required for functionality. A substitution of W67F is shown to be accompanied by the L105W substitution. Molecular dynamics shows that the W67 binds to the substrate via a {pi}-{pi} stacking and a salt bridge, the latter being stronger in a virtual W67F/L105W double mutant of the resolved structure of Scytalido-carboxyl peptidase-B (PDB ID: 2IFW)). Additional likely fatal mutants are discussed.\n\nUpon sequence scrutiny we obtained a set of 233 sequences that in all likelihood lack false positives. This was used to reconstruct a Bayesian phylogenetic tree. We identified 14 putative specificity determining positions (SDPs) of which four are explained by mere structural explanations and nine seem to correspond to functional diversification related wit substrate binding ans specificity. A first sub-network of SDPs is related to substrate specificity whereas the second sub-network seems to affect the dynamics of three loops that are involved in substrate binding.\n\nHighlightsEqolisins are acid proteases found in prokaryotes and fungi only.\n\nThe recently co-evolved W67F-L105W substitutions promote substrate binding\n\nTwo Specificity Determining Networks, SDN1 and 2, were identified\n\nSDN1 has four Specificity Determining Positions involved in substrate specificity\n\nSDN2 has five Specificity Determining Positions involved in loop-substrate dynamics