Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target

Sarah C. Atkinson, Con Dogovski, Kathleen Wood, Michael D.W. Griffin, Michael A. Gorman, Lilian Hor, Cyril F. Reboul, Ashley M. Buckle, Joachim Wuttke, Michael W. Parker, Renwick C.J. Dobson, Matthew A. Perugini

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Protein dynamics manifested through structural flexibility play a central role in the function of biological molecules. Here we explore the substrate-mediated change in protein flexibility of an antibiotic target enzyme, Clostridium botulinum dihydrodipicolinate synthase. We demonstrate that the substrate, pyruvate, stabilizes the more active dimer-of-dimers or tetrameric form. Surprisingly, there is little difference between the crystal structures of apo and substrate-bound enzyme, suggesting protein dynamics may be important. Neutron and small-angle X-ray scattering experiments were used to probe substrate-induced dynamics on the sub-second timescale, but no significant changes were observed. We therefore developed a simple technique, coined protein dynamics-mass spectrometry (ProD-MS), which enables measurement of time-dependent alkylation of cysteine residues. ProD-MS together with X-ray crystallography and analytical ultracentrifugation analyses indicates that pyruvate locks the conformation of the dimer that promotes docking to the more active tetrameric form, offering insight into ligand-mediated stabilization of multimeric enzymes. Atkinson et al. show that pyruvate binding locks the conformation of the C. botulinum DHDPS dimer that promotes tetramerization. They describe a new method (ProD-MS) that assesses protein dynamics on a slow (second-minute) timescale.

Original languageEnglish
Pages (from-to)948-959
Number of pages12
JournalStructure
Volume26
Issue number7
DOIs
Publication statusPublished - 3 Jul 2018

Keywords

  • analytical ultracentrifugation
  • diaminopimelate
  • dihydrodipicolinate synthase
  • enzyme
  • lysine
  • mass spectrometry
  • ProD-MS
  • protein dynamics
  • slow dynamics
  • X-ray

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