Characterization of the Interaction between Arginine Methyltransferase Hmt1 and Its Substrate Npl3: Use of Multiple Cross-Linkers, Mass Spectrometric Approaches, and Software Platforms

Anal Chem. 2018 Aug 7;90(15):9101-9108. doi: 10.1021/acs.analchem.8b01525. Epub 2018 Jul 25.

Abstract

This study investigated the enzyme-substrate interaction between Saccharomyces cerevisiae arginine methyltransferase Hmt1p and nucleolar protein Npl3p, using chemical cross linking/mass spectrometry (XL/MS). We show that XL/MS can capture transient interprotein interactions that occur during the process of methylation, involving a disordered region in Npl3p with tandem SRGG repeats, and we confirm that Hmt1p and Npl3p exist as homomultimers. Additionally, the study investigated the interdependencies between variables of an XL/MS experiment that lead to the identification of identical or different cross-linked peptides. We report that there are substantial benefits, in terms of biologically relevant cross-links identified, that result from the use of two mass-spectrometry-cleavable cross-linkers [disuccinimido sulfoxide (DSSO) and disuccinimido dibutyric urea (DSBU)], two fragmentation approaches [collision-induced dissociation and electron-transfer dissociation (CID+ETD)] and stepped high-energy collision dissociation (HCD)], and two programs (MeroX and XlinkX). We also show that there are specific combinations of XL/MS methods that are more successful than others for the two proteins investigated here; these are explored in detail in the text. Data are available via ProteomeXchange with identifier PXD008348.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cross-Linking Reagents / chemistry
  • Cross-Linking Reagents / metabolism*
  • Molecular Docking Simulation
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / metabolism*
  • Protein Conformation
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping / methods*
  • Protein Interaction Maps
  • Protein Multimerization
  • Protein-Arginine N-Methyltransferases / chemistry
  • Protein-Arginine N-Methyltransferases / metabolism*
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / metabolism*
  • Repressor Proteins / chemistry
  • Repressor Proteins / metabolism*
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Software
  • Tandem Mass Spectrometry / methods*

Substances

  • Cross-Linking Reagents
  • NPL3 protein, S cerevisiae
  • Nuclear Proteins
  • RNA-Binding Proteins
  • Repressor Proteins
  • Saccharomyces cerevisiae Proteins
  • HMT1 protein, S cerevisiae
  • Protein-Arginine N-Methyltransferases