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

Cite this

Atkinson, S. C., Dogovski, C., Wood, K., Griffin, M. D. W., Gorman, M. A., Hor, L., ... Perugini, M. A. (2018). Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target. Structure, 26(7), 948-959. https://doi.org/10.1016/j.str.2018.04.014
Atkinson, Sarah C. ; Dogovski, Con ; Wood, Kathleen ; Griffin, Michael D.W. ; Gorman, Michael A. ; Hor, Lilian ; Reboul, Cyril F. ; Buckle, Ashley M. ; Wuttke, Joachim ; Parker, Michael W. ; Dobson, Renwick C.J. ; Perugini, Matthew A. / Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target. In: Structure. 2018 ; Vol. 26, No. 7. pp. 948-959.
@article{630ae8a007534a93af7c851ddc37838d,
title = "Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target",
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.",
keywords = "analytical ultracentrifugation, diaminopimelate, dihydrodipicolinate synthase, enzyme, lysine, mass spectrometry, ProD-MS, protein dynamics, slow dynamics, X-ray",
author = "Atkinson, {Sarah C.} and Con Dogovski and Kathleen Wood and Griffin, {Michael D.W.} and Gorman, {Michael A.} and Lilian Hor and Reboul, {Cyril F.} and Buckle, {Ashley M.} and Joachim Wuttke and Parker, {Michael W.} and Dobson, {Renwick C.J.} and Perugini, {Matthew A.}",
year = "2018",
month = "7",
day = "3",
doi = "10.1016/j.str.2018.04.014",
language = "English",
volume = "26",
pages = "948--959",
journal = "Structure",
issn = "0969-2126",
publisher = "Elsevier",
number = "7",

}

Atkinson, SC, Dogovski, C, Wood, K, Griffin, MDW, Gorman, MA, Hor, L, Reboul, CF, Buckle, AM, Wuttke, J, Parker, MW, Dobson, RCJ & Perugini, MA 2018, 'Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target' Structure, vol. 26, no. 7, pp. 948-959. https://doi.org/10.1016/j.str.2018.04.014

Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target. / Atkinson, Sarah C.; Dogovski, Con; Wood, Kathleen; Griffin, Michael D.W.; Gorman, Michael A.; Hor, Lilian; Reboul, Cyril F.; Buckle, Ashley M.; Wuttke, Joachim; Parker, Michael W.; Dobson, Renwick C.J.; Perugini, Matthew A.

In: Structure, Vol. 26, No. 7, 03.07.2018, p. 948-959.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target

AU - Atkinson, Sarah C.

AU - Dogovski, Con

AU - Wood, Kathleen

AU - Griffin, Michael D.W.

AU - Gorman, Michael A.

AU - Hor, Lilian

AU - Reboul, Cyril F.

AU - Buckle, Ashley M.

AU - Wuttke, Joachim

AU - Parker, Michael W.

AU - Dobson, Renwick C.J.

AU - Perugini, Matthew A.

PY - 2018/7/3

Y1 - 2018/7/3

N2 - 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.

AB - 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.

KW - analytical ultracentrifugation

KW - diaminopimelate

KW - dihydrodipicolinate synthase

KW - enzyme

KW - lysine

KW - mass spectrometry

KW - ProD-MS

KW - protein dynamics

KW - slow dynamics

KW - X-ray

UR - http://www.scopus.com/inward/record.url?scp=85046681953&partnerID=8YFLogxK

U2 - 10.1016/j.str.2018.04.014

DO - 10.1016/j.str.2018.04.014

M3 - Article

VL - 26

SP - 948

EP - 959

JO - Structure

JF - Structure

SN - 0969-2126

IS - 7

ER -