title: Nepenthesin from monkey cups for hydrogen/deuterium exchange mass spectrometry.
abstract: 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 digestion 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 disordered regions. In this study we present nepenthesin-containing secretions of the pitcher plant Nepenthes, commonly 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 selectivity 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 analysis with no detectable autolysis, avoiding the complication 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 analysis of protein complexes in the nonhomologous end-joining DNA repair pathway. The analysis of XRCC4 binding 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 workflow. 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 phosphoregulated. 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 binding partners.
authors.affiliation: Department of Biochemistry and Molecular Biology and the Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Martial
authors.initials: M
authors.lastname: Rey
authors.forename: Menglin
authors.initials: M
authors.lastname: Yang
authors.forename: Kyle M
authors.initials: KM
authors.lastname: Burns
authors.forename: Yaping
authors.initials: Y
authors.lastname: Yu
authors.forename: Susan P
authors.initials: SP
authors.lastname: Lees-Miller
authors.forename: David C
authors.initials: DC
authors.lastname: Schriemer
mesh_terms: D059766:DNA End-Joining Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry; D041961:Deuterium Exchange Measurement / Q000379:methods*; D006859:Hydrogen / Q000737:chemistry; D019684:Magnoliopsida / Q000737:chemistry / Q000201:enzymology*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010434:Pepsin A / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; C101467:XRCC4 protein, human; D006859:Hydrogen; D003903:Deuterium; C111164:BRAP protein, human; D044767:Ubiquitin-Protein Ligases; D010447:Peptide Hydrolases; D010434:Pepsin A