Nepenthesin from Monkey Cups for Hydrogen/ Deuterium Exchange Mass Spectrometry*□S

Studies of protein dynamics, structure and interactions using hydrogen/deuterium exchange mass spectrometry (HDX-MS) have sharply increased over the past 5–10 years. The predominant technology requires fast diges-tion at pH 2–3 to retain deuterium label. Pepsin is used almost exclusively, but it provides relatively low efficiency under the constraints of the experiment, and a selectivity profile that renders poor coverage of intrinsically disor-dered regions. In this study we present nepenthesin-con-taining secretions of the pitcher plant Nepenthes, com-monly called monkey cups, for use in HDX-MS. We show that nepenthesin is at least 1400-fold more efficient than pepsin under HDX-competent conditions, with a selectiv-ity profile that mimics pepsin in part, but also includes efficient cleavage C-terminal to “forbidden ” residues K, R, H, and P. High efficiency permits a solution-based analy-sis with no detectable autolysis, avoiding the complica-tion of immobilized enzyme reactors. Relaxed selectivity promotes high coverage of disordered regions and the ability to “tune ” the mass map for regions of interest. Nepenthesin-enriched secretions were applied to an anal-ysis of protein complexes in the nonhomologous end-joining DNA repair pathway. The analysis of XRCC4 bind-ing to the BRCT domains of Ligase IV points to secondary interactions between the disordered C-terminal tail of XRCC4 and remote regions of the BRCT domains, which could only be identified with a nepenthesin-based work-flow. HDX data suggest that stalk-binding to XRCC4 primes a BRCT conformation in these remote regions to support tail interaction, an event which may be phospho-regulated. We conclude that nepenthesin is an effective alternative to pepsin for all HDX-MS applications, and especially for the analysis of structural transitions among intrinsically disordered proteins and their bindin