title: 3D Structure Determination of an Unstable Transient Enzyme Intermediate by Paramagnetic NMR Spectroscopy.
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.
authors.affiliation: State Key Laboratory of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
authors.forename: Jia-Liang
authors.initials: JL
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
authors.forename: Xiao
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China.
authors.forename: Chan
authors.initials: C
authors.lastname: Cao
authors.affiliation: Research School of Chemistry, Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: Gottfried
authors.initials: G
authors.lastname: Otting
authors.affiliation: State Key Laboratory of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin, 300071, China. xunchengsu@nankai.edu.cn.
authors.forename: Xun-Cheng
authors.identifier: 0000-0003-3051-0047
authors.initials: XC
authors.lastname: Su
mesh_terms: D019881:Aminoacyltransferases / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D013211:Staphylococcus aureus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases
keywords: NMR spectroscopy; enzyme catalysis; protein dynamics; protein structures; transient intermediates