Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target.
Structure 2018 Jul 03; 26(7):948-959.e5.

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.

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Authors+Show Affiliations

Atkinson SCDepartment of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia; Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, University of Melbourne, Parkville, VIC 3010, Australia.
Dogovski CDepartment of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, University of Melbourne, Parkville, VIC 3010, Australia.
Wood KAustralian Nuclear Science and Technology Organisation, Lucas Heights, NSW 2234, Australia.
Griffin MDWDepartment of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, University of Melbourne, Parkville, VIC 3010, Australia.
Gorman MAACRF Rational Drug Discovery Centre, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia.
Hor LDepartment of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, University of Melbourne, Parkville, VIC 3010, Australia; Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia; ARC Centre of Excellence in Advanced Molecular Imaging, La Trobe University, Melbourne, VIC 3086, Australia.
Reboul CFDepartment of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.
Buckle AMDepartment of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia.
Wuttke JJuelich Centre for Neutron Science (JCNS), at Heinz Maier-Leibnitz Zentrum (MLZ), Forschungszentrum Juelich GmbH, Lichtenstrasse 1, Garching 85 747, Germany.
Parker MWDepartment of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, University of Melbourne, Parkville, VIC 3010, Australia; ACRF Rational Drug Discovery Centre, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia.
Dobson RCJDepartment of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, University of Melbourne, Parkville, VIC 3010, Australia; Biomolecular Interaction Centre, School of Biological Sciences, University of Canterbury, Private Bag, Christchurch 4800, New Zealand.
Perugini MADepartment of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, 30 Flemington Road, University of Melbourne, Parkville, VIC 3010, Australia; Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC 3086, Australia. Electronic address: m.perugini@latrobe.edu.au.

MeSH

AlkylationBacterial ProteinsClostridium botulinumCrystallography, X-RayCysteineEnzyme StabilityHydro-LyasesModels, MolecularProtein ConformationProtein MultimerizationPyruvic AcidScattering, Small AngleX-Ray Diffraction

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

29804823