Transmembrane Substrate Unfolding In Intramembrane Proteolysis

In recent years impressive progress has been made in our understanding of intramembrane cleaving proteases (iCliPs), enzymes that carry out proteolytic reactions within cell membranes, and their substrates, but basic biochemical questions such as what determines a transmembrane helix to be a substrate and how cleavage site is dictated remain unresolved. Much onus as to why we sill do not understand substrate specificity in these enzymes falls to the difficulties in monitoring enzyme-substrate interactions in a lipid or membrane-mimetic environment. Here we will present deep-UV resonance Raman spectroscopy data describing discrete structural characteristics of substrates prior to and during interactions with iCLiPS indicating structural flexibility in both environments is an intrinsic property of cleavable substrates. Furthermore, we also will present evidence that mutagenesis of substrates that abolishes intramembrane proteolysis do not significantly influence binding and dissociation constants of enzyme and substrate, while they do convey distinct changes to the intrinsic structural flexibility of the substrate. The mutations also alter the extent to which the enzyme influences the substrate structure during binding. Overall these results imply a synergy between a sequence complementarity between enzyme and substrate, which requires adequate structural flexibility of the substrate transmembrane helical sequence in order to expose the peptide bond for cleavage.