Probing activation‐driven changes in coagulation factor IX by mass spectrometry

BACKGROUND: Activated factor IX (FIXa) is an inefficient enzyme that needs activated factor VIII (FVIII) for full activity. Recently, we identified a network of FVIII‐driven changes in FIXa employing hydrogen‐deuterium eXchange mass spectrometry (HDX‐MS). Some changes also occurred in active‐site inhibited FIXa, but others were not cofactor‐driven, in particular those within the 220‐loop (in chymotrypsin numbering). OBJECTIVE: The aim of this work is to better understand the zymogen‐to‐enzyme transition in FIX, with specific focus on substrate‐driven changes at the catalytic site. METHODS: Footprinting mass spectrometry by HDX and Tandem‐Mass Tags (TMT) labelling were used to explore changes occurring upon the conversion from FIX into FIXa. Mutagenesis and kinetic studies served to assess the role of the 220‐loop. RESULTS: HDX‐MS displayed remarkably few differences between FIX and FIXa. In comparison with FIX, FIXa did exhibit decreased deuterium uptake at the N‐terminus region. This was more prominent when the FIXa active site was occupied by an irreversible inhibitor. TMT‐labelling showed that the N‐terminus is largely protected from labelling, and that inhibitor binding increases protection to a minor extent. Occupation of the active site also reduced deuterium uptake within the 220‐loop backbone. Mutagenesis within the 220‐loop revealed that a putative H‐bond network contributes to FIXa activity. TMT labeling of the N‐terminus suggested that these 220‐loop variants are more zymogen‐like than wild‐type FIXa. CONCLUSION: In the absence of cofactor and substrate, FIXa is predominantly zymogen‐like. Stabilization in its enzyme‐like form involves, apart from FVIII‐binding, also interplay between the 220‐loop, N‐terminus, and the substrate binding site.