Refining the Structural Model of a HeterohexamericProtein Complex: Surface Induced Dissociation and Ion Mobility ProvideKey Connectivity and Topology Information

摘要

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 intersubunitcontacts and relative interfacial strengths are obtained. The resultsshow that TNH is a dimer of αβγ trimers, that withinthe trimer the α, β subunits bind most strongly, and thatthe primary contact between the two trimers is through a γ–γinterface. Collisional cross sections (CCSs) measured from IM experimentsare used as constraints for postulating the arrangement of the subunitsrepresented by coarse-grained spheres. Covalent labeling (surfacemapping) together with protein complex homology modeling and dockingof trimers to form hexamer are utilized with all the above informationto propose the likely quaternary structure of TNH, with chemical cross-linkingproviding cross-links consistent with the proposed structure. Thenovel feature of this approach is the use of SID-MS with ion mobilityto define complete connectivity and relative interfacial areas ofa heterohexameric protein complex, providing much more informationthan is available from solution disruption. That information, whencombined with CCS-guided coarse-grained modeling and covalent labelingrestraints for homology modeling and trimer–trimer docking,provides atomic models of a previously uncharacterized heterohexamericprotein complex.