3D Structure Determination of an Unstable Transient Enzyme Intermediate by Paramagnetic NMR Spectroscopy

Angew Chem Int Ed Engl. 2016 Oct 24;55(44):13744-13748. doi: 10.1002/anie.201606223. Epub 2016 Oct 4.

Abstract

Enzyme catalysis relies on conformational plasticity, but structural information on transient intermediates is difficult to obtain. We show that the three-dimensional (3D) structure of an unstable, low-abundance enzymatic intermediate can be determined by nuclear magnetic resonance (NMR) spectroscopy. The approach is demonstrated for Staphylococcus aureus sortase A (SrtA), which is an established drug target and biotechnological reagent. SrtA is a transpeptidase that converts an amide bond of a substrate peptide into a thioester. By measuring pseudocontact shifts (PCSs) generated by a site-specific cysteine-reactive paramagnetic tag that does not react with the active-site residue Cys184, a sufficient number of restraints were collected to determine the 3D structure of the unstable thioester intermediate of SrtA that is present only as a minor species under non-equilibrium conditions. The 3D structure reveals structural changes that protect the thioester intermediate against hydrolysis.

Keywords: NMR spectroscopy; enzyme catalysis; protein dynamics; protein structures; transient intermediates.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminoacyltransferases / chemistry*
  • Aminoacyltransferases / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism
  • Cysteine Endopeptidases / chemistry*
  • Cysteine Endopeptidases / metabolism
  • Models, Molecular
  • Molecular Structure
  • Nuclear Magnetic Resonance, Biomolecular*
  • Protein Conformation
  • Staphylococcus aureus / enzymology*

Substances

  • Bacterial Proteins
  • Aminoacyltransferases
  • sortase A
  • Cysteine Endopeptidases