Molecular dynamics and structure function analysis show that substrate binding and specificity are 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 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.Electronic supplementary materialThe online version of this article (10.1186/s12859-018-2348-2) contains supplementary material, which is available to authorized users.

Molecular and in vivo studies of a glutamate-class prolyl-endopeptidase for coeliac disease therapy

“…The glutamate is assisted by a glutamine (Q 107 in SCP-B), hence the family name eq olysins 55 . These residues are invariant within the family and are flanked by very similar residues 66 , 67 .…”

“…However, there are differences in the size of the PD and the CD, the active-site environment, the substrate-binding modus and specificity, as well as the chemical mechanism of catalysis. Furthermore, whereas eqolysins are restricted to fungi and bacteria 67 , 73 , potential neprosin orthologues with ~35–40% sequence identity are widely found in (and restricted to) plants, including gluten-containing crops. This suggests the neprosin family may have originated from a SCP-B ancestor by horizontal gene transfer from a bacterium or fungus to a plant, as previously described for other proteins 74 .…”

Overview of the Properties of Glutamic Peptidases That Are Present in Plant and Bacterial Pathogens and Play a Role in Celiac Disease and Cancer

“…In 2018, Stocchi et al reported that substrate binding and specificity are major forces in the functional diversification of eqolisins. 51 2.2. Aspergilloglutamic Peptidase.…”

“…The eqolisins form a small superfamily of acid proteases that includes many paralogs in acidic fungi. In 2018, Stocchi et al reported that substrate binding and specificity are major forces in the functional diversification of eqolisins …”

Comprehensive characterization of the complex BAHD acyltransferase family from 218 land plants species: phylogenomic analysis and identification of specificity determinant positions

“…We apply this using a network approach and a network connectivity score in order to select the most probable SDPs. We have ap pC lied this approach to the identification of SDPs in other families [19] [20].…”