Functional Diversification of Tripeptidyl Peptidase and Endopeptidase Sedolisins in Fungi

Sedolisins are acid proteases that are related to the basic subtilisins. They have been identified in all three superkingdoms but are not ubiquitous, although fungi that secrete acids as part of their lifestyle can have up to six paralogs. Both tripeptidyl peptidase (TPP) and endopeptidase activity have been identified and it has been suggested that these correspond to separate subfamilies.\n\nWe studied eukaryotic sedolisins by computational analysis. A maximum likelihood tree shows three major clades of which two contain only fungal sequences. One fungal clade contains all known TPPs whereas the other contains the endosedolisins. We identified four cluster specific inserts (CSIs) in endosedolisins, of which CSIs 1, 3 and 4 appear as solvent exposed according to structure modeling. Part of CSI2 is exposed but a short stretch forms a novel and partially buried -helix that induces a conformational change near the binding pocket. We also identified a total of 12 specificity determining positions (SDPs) divided over three SDP sub-networks. The major SDP network contains eight directly connected SDPs and modeling of virtual mutants suggests a key role for the W307A or F307A substitution. This substitution is accompanied by a group of four SDPs that physically interact at the interface of the catalytic domain and the enzymes prosegment. Modeling of virtual mutants suggests these SDPs are indeed required to compensate the conformational change induced by CSI2 and the A307. The additional major network SDPs as well as the two small SDP networks appear to be linked to this major substitution, all together explaining the hypothesized functional diversification of fungal sedolisins.\n\nHighlightsThere are two sedolisin subfamilies in fungi: tripeptidyl peptidases and endopeptidases\nFunctional Diversification of fungal sedolisins led to a conformational change in the pocket\nFunctional Diversification centers around buried SDP307\nSDP307 is aromatic in TPPs and Alanine in endosedolisins\nAdditional SDPs affect the interaction between core and chaperone-like prosegment