title: Refining the Structural Model of a Heterohexameric Protein Complex: Surface Induced Dissociation and Ion Mobility Provide Key Connectivity and Topology Information.
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.
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University , Columbus, Ohio 43210, United States.
authors.forename: Yang
authors.initials: Y
authors.lastname: Song
authors.affiliation: Department of Chemistry and Biochemistry, The University of Arizona, Tucson, Arizona 85721, United States; Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States.
authors.forename: Micah T
authors.initials: MT
authors.lastname: Nelp
authors.affiliation: Department of Chemistry and Biochemistry, The University of Arizona, Tucson, Arizona 85721, United States; Department of Chemistry, University of Utah, Salt Lake City, Utah 84112, United States.
authors.forename: Vahe
authors.initials: V
authors.lastname: Bandarian
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University , Columbus, Ohio 43210, United States.
authors.forename: Vicki H
authors.initials: VH
authors.lastname: Wysocki