Investigation of Anthranilate Synthase Structures by Mass Spectrometry
Structural Biology
Final Report Abstract
Currently, most structural information on protein complexes is derived from X-ray crystallographic data. However, only a rather small number of protein complexes have been crystallized so far. Furthermore, crystallization conditions can result in the perturbation of physiological relevant protein-protein interactions and inevitably result in the formation of (artificial) crystal contracts. Newly formed crystal contacts and physiologically-relevant proteinprotein interactions co-exist, which can lead to ambiguity in the annotation of quaternary structures. Aim of this work was to establish mass spectrometry based structural determination methods that are generally applicable and particularly useful to determine the quaternary structure variability within groups of homologous protein complexes. The quaternary structures of tryptophan synthase and anthranilate synthase homologs were probed by native mass spectrometry as well as cross-linking bottom-up mass spectrometry. All complexes were determined to be heterotetramers. Fragmentation by surface-induced dissociation after selection resulted in the generation of characteristic sub-complexes that provided information on the initial structure of those complexes. The dissociation of all +18 tryptophan synthases by SID resulted in the generation of the following sub-complexes: A, AB, ABB and BB, consistent with a conserved ABBA quaternary structure. Dissociation of +19 anthranilate synthases resulted in the generation of E-, G-, EG-, and EGG-sub-complexes for the S. solfataricus ANS and G-, E-, EE, GEE-sub-complexes for the S. typhimurium ANS. This suggests that these two ANS homologs differ in their quaternary structures with only the G-subunits of S. solfataricus interacting with each other within the complex. Further support for the unusual EGGE S. solfataricus structure was obtained from cross-linking experiments with several cross-links being identified between the G-subunits. Thus, native MS in combination with cross-linking MS provides the tools to screen for quaternary structure variation of homologous protein complexes.
Publications
- Nucleic Acids Res 2017, 45, 11867-11877
M. S. Park, H. D. Phan, F. Busch, S. H. Hinckley, J. A. Brackbill, V. H. Wysocki, K. Nakanishi
(See online at https://doi.org/10.1093/nar/gkx916)
