Probing Enzyme-Substrate Complex Structural Dynamics during Intramembrane Proteolysis

Understanding how intramembrane proteolysis is carried out and controlled is important for a variety of reasons, not the least of which is its implied importance in Alzheimer's disease onset/progression. One of the most challenging questions in this field lies in identifying how substrates, single transmembrane helical domains (or type-I,II,III membrane proteins), are recognized by the intramembrane-cleaving protease (I-CLiP) complexes in the absence of discernible sequence motifs. Until this is understood, how cleavage sites are defined by the substrate-enzyme interaction will remain a mystery. Of course, this is the big question, as diseases like Alzheimer's are referred to be resultant from “mis-cleavage”, which is manifested by promiscuity of cleavage site choice by the enzyme. One hypothesis for substrate recognition/cleavage site choice that has gained broader acceptance is that specific substrate structural perturbations during enzyme-substrate interactions differentiate cleaved from non-cleaved single span helical domains. Here we present our findings demonstrating the structural dependence of substrate sequences to potentially helix breaking residue content during their interactions with various I-CLiPs. Interestingly, using a combination of isotopic labeling/exchange coupled to UV-resonance Raman we are able to characterize subtle structural perturbations during the protein-protein interactions within a membrane as well as define the types and extent of non α-helical structures explored by the bound substrate.