Molecular Dynamics and Structure Function Analysis show that Substrate Binding and Specificity are major Forces in the functional Diversification of Eqolisins

N. STOCCHI; M. V. REVUELTA; P. A. LANZA CASTRONUOVO; D. M. A. VERA; A. TEN HAVE

BMC BIOINFORMATICS

BIOMED CENTRAL LTD

Lugar: Londres; Año: 2018 vol. 19 p. 1 - 16

1471-2105

Background:Eqolisins are rare acid proteases found in archaea, bacteria andfungi. Certain fungi secrete acids as part of their lifestyle andinterestingly these also have many eqolisin paralogs, up to nineparalogs have been recorded. This suggests a process of functionalredundancy and diversification has occurred, which was the subject ofthe research we performed and describe here.Results:We identified eqolisin homologs by means of iterative HMMER analysisof the NR database. The identified sequences were scrutinized forwhich new hallmarks were identified by molecular dynamics simulationsof mutants in highly conserved positions, using the structure of aneqolisin that was crystallized in the presence of a transition stateinhibitor. Four conserved glycines were shown to be important forfunctionality. A substitution of W67F is shown to be accompanied bythe L105W substitution. Molecular dynamics shows that the W67 bindsto the substrate via a π-πstacking and a salt bridge, the latter being stronger in a virtualW67F/L105W double mutant of the resolved structure ofScytalido-carboxyl peptidase-B (PDB ID: 2IFW). Additional problematicmutations are discussed. Upon sequence scrutiny we obtained a set of233 sequences that was used to reconstruct a Bayesian phylogenetictree. We identified 14 putative specificity determining positions(SDPs) of whichfour are explained by mere structural explanations and nine seem tocorrespond to functional diversification related with substratebinding and specificity. A first sub-network of SDPs is related tosubstrate specificity whereas the second sub-network seems to affectthe dynamics of three loops that are involved in substrate binding.Conclusion:Theeqolisins form a small superfamily of acid proteases withnevertheless many paralogs in acidic fungi. Functional redundancy hasresulted in diversification related to substrate specificity andsubstrate bindingp { margin-bottom: 0cm; direction: ltr; color: rgb(0, 0, 0); line-height: 200%; text-align: justify; }p.western { font-family: "Times New Roman", serif; font-size: 12pt; }p.cjk { font-family: "Droid Sans"; font-size: 12pt; }p.ctl { font-family: "Times New Roman", serif; font-size: 12pt; }a.western:visited { }a.cjk:link { }a.ctl:link { }