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

Highlights

• Crystal structure of C. botulinum DHDPS was solved in apo and substrate-bound form

• C. botulinum DHDPS has reduced protein dynamics when bound to its substrate

• Substrate binding locks the conformation of the dimer that promotes tetramerization

• New ProD-MS method that assesses protein dynamics on a slow timescale is described

Summary

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.