Refining the structural model of a heterohexameric protein complex: Surface induced dissociation and ion mobility provide key connectivity and topology information

Yang Song, Micah T. Nelp, Vahe Bandarian, Vicki H. Wysocki

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Toyocamycin nitrile hydratase (TNH) is a protein hexamer that catalyzes the hydration of toyocamycin to produce sangivamycin. The structure of hexameric TNH and the arrangement of subunits within the complex, however, have not been solved by NMR or X-ray crystallography. Native mass spectrometry (MS) clearly shows that TNH is composed of two copies each of the α, β, and γ subunits. Previous surface induced dissociation (SID) tandem mass spectrometry on a quadrupole time-of-flight (QTOF) platform suggests that the TNH hexamer is a dimer composed of two αβγ trimers; furthermore, the results suggest that α-β interact most strongly (Blackwell et al. Anal. Chem. 2011, 83, 2862-2865). Here, multiple complementary MS based approaches and homology modeling have been applied to refine the structure of TNH. Solution-phase organic solvent disruption coupled with native MS agrees with the previous SID results. By coupling surface induced dissociation with ion mobility mass spectrometry (SID/IM), further information on the intersubunit contacts and relative interfacial strengths are obtained. The results show that TNH is a dimer of αβγ trimers, that within the trimer the α, β subunits bind most strongly, and that the primary contact between the two trimers is through a γ-γ interface. Collisional cross sections (CCSs) measured from IM experiments are used as constraints for postulating the arrangement of the subunits represented by coarse-grained spheres. Covalent labeling (surface mapping) together with protein complex homology modeling and docking of trimers to form hexamer are utilized with all the above information to propose the likely quaternary structure of TNH, with chemical cross-linking providing cross-links consistent with the proposed structure. The novel feature of this approach is the use of SID-MS with ion mobility to define complete connectivity and relative interfacial areas of a heterohexameric protein complex, providing much more information than is available from solution disruption. That information, when combined with CCS-guided coarse-grained modeling and covalent labeling restraints for homology modeling and trimer-trimer docking, provides atomic models of a previously uncharacterized heterohexameric protein complex.

Original languageEnglish (US)
Pages (from-to)477-487
Number of pages11
JournalACS Central Science
Volume1
Issue number9
DOIs
StatePublished - Dec 23 2015

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Toyocamycin
Refining
Mass spectrometry
Topology
Ions
Proteins
Dimers
Labeling
sangivamycin
X ray crystallography
Hydration
Organic solvents
nitrile hydratase
Nuclear magnetic resonance

ASJC Scopus subject areas

  • Chemical Engineering(all)
  • Chemistry(all)

Cite this

Refining the structural model of a heterohexameric protein complex : Surface induced dissociation and ion mobility provide key connectivity and topology information. / Song, Yang; Nelp, Micah T.; Bandarian, Vahe; Wysocki, Vicki H.

In: ACS Central Science, Vol. 1, No. 9, 23.12.2015, p. 477-487.

Research output: Contribution to journalArticle

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abstract = "Toyocamycin nitrile hydratase (TNH) is a protein hexamer that catalyzes the hydration of toyocamycin to produce sangivamycin. The structure of hexameric TNH and the arrangement of subunits within the complex, however, have not been solved by NMR or X-ray crystallography. Native mass spectrometry (MS) clearly shows that TNH is composed of two copies each of the α, β, and γ subunits. Previous surface induced dissociation (SID) tandem mass spectrometry on a quadrupole time-of-flight (QTOF) platform suggests that the TNH hexamer is a dimer composed of two αβγ trimers; furthermore, the results suggest that α-β interact most strongly (Blackwell et al. Anal. Chem. 2011, 83, 2862-2865). Here, multiple complementary MS based approaches and homology modeling have been applied to refine the structure of TNH. Solution-phase organic solvent disruption coupled with native MS agrees with the previous SID results. By coupling surface induced dissociation with ion mobility mass spectrometry (SID/IM), further information on the intersubunit contacts and relative interfacial strengths are obtained. The results show that TNH is a dimer of αβγ trimers, that within the trimer the α, β subunits bind most strongly, and that the primary contact between the two trimers is through a γ-γ interface. Collisional cross sections (CCSs) measured from IM experiments are used as constraints for postulating the arrangement of the subunits represented by coarse-grained spheres. Covalent labeling (surface mapping) together with protein complex homology modeling and docking of trimers to form hexamer are utilized with all the above information to propose the likely quaternary structure of TNH, with chemical cross-linking providing cross-links consistent with the proposed structure. The novel feature of this approach is the use of SID-MS with ion mobility to define complete connectivity and relative interfacial areas of a heterohexameric protein complex, providing much more information than is available from solution disruption. That information, when combined with CCS-guided coarse-grained modeling and covalent labeling restraints for homology modeling and trimer-trimer docking, provides atomic models of a previously uncharacterized heterohexameric protein complex.",
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