Molecular dynamics and structure function analysis show that substrate binding and specificity are major forces in the functional diversification of Eqolisins

Nicolás Stocchi,Priscila Ailín Lanza Castronuovo,D Mariano A Vera,María Victoria Revuelta,Arjen Ten Have

doi:10.1186/s12859-018-2348-2

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 a process of functional redundancy and diversification has occurred, which was the subject of the research we performed and describe here.ResultsWe identified eqolisin homologs by means of iterative HMMER analysis of the NR database. The identified sequences were scrutinized for which new hallmarks were identified by molecular dynamics simulations of mutants in 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 important 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 π-π 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 problematic mutations are discussed. Upon sequence scrutiny we obtained a set of 233 sequences that 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 with substrate binding and 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.ConclusionThe eqolisins form a small superfamily of acid proteases with nevertheless many paralogs in acidic fungi. Functional redundancy has resulted in diversification related to substrate specificity and substrate binding.

Highlights

Eqolisins are rare acid proteases found in archaea, bacteria and fungi
A complete preliminary Multiple Sequence Aignments (MSA) with all identified sequences can be found in Additional file 2
The sensitive HMMER search resulted in a collection of homologous sequences that may include sequences from non-functional homologs (NFHs) or incorrectly predicted gene models

Summary

Introduction

Eqolisins are rare acid proteases found in archaea, bacteria and fungi. Certain fungi secrete acids as part of their lifestyle and interestingly these have many eqolisin paralogs, up to nine paralogs have been recorded. Acid proteases The three major families of acid or carboxyl peptidases recognized by MEROPS [1] are the well studied, pepstatin-sensitive, eukaryotic aspartic proteinases (A01, APs, for review see [2]), part of the aspartic proteinase clan A; the more recently identified sedolisins (S53) and the novel eqolisins or glutamic peptidases (G01), both recently reviewed [3]. Both sedolisins and eqolisins were first thought to be pepstatin-insensitive AP variants but the structures that were resolved showed they are unrelated. Other characterized eqolisins are Aspergillus niger carboxyl peptidase (ANCP) [10], which 3D structure has been resolved (1Y43) [11], Sclerotinia sclerotiorum carboxyl peptidase [12], Cryphonectria parasitica peptidases B and C [13], Talaromyces emersonii carboxyl peptidase TGP1 [14] and bacterial Alicyclobacillus sp. pepG1 [5]

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Conclusion