Abstract
Biotin protein ligase (BPL) catalyzes the covalent attachment of biotin onto biotin-dependent enzymes, where it functions as an essential cofactor. Eukaryotic BPLs are distinct due to the presence of a large N-terminal extension to the conserved catalytic domain and C-terminal cap. No high-resolution structures of a eukaryotic BPL have been solved; however, previous functional studies revealed the N-terminal extension interacts with the biotinylation substrate. Mass spectrometry (MS) and complementary techniques were utilized to investigate the structure of the yeast Saccharomyces cerevisiae BPL (ScBPL). Lower resolution techniques suggested holo-ScBPL had a more compact structure and sampled fewer conformational states. In addition, solution-phase and a charge state dependent gas-phase stabilization was observed. Hydrogen-deuterium exchange (HDX) MS provided experimental validation of the AlphaFold predicted structure of ScBPL, with a folded domain structurally homologous to a glutamine amidotransferase identified in the N-terminal extension, and a mostly homologous catalytic domain to that of other species' BPLs. Further HDX analyses identified localized conformational changes in the ScBPL active site and N-terminal domain that occur concomitantly with ligand binding. These data provide novel insights into the unique structure of a class III BPL and how ligands influence this structure for catalysis of protein biotinylation.