Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target Academic Article uri icon

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.

authors

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

publication date

  • 2018