Dual roles of F123 in protein homodimerization and inhibitor binding to biotin protein ligase from Staphylococcus aureus

作者：da Costa Tatiana P Soares; Yap Min Y; Perugini Matthew A; Wallace John C; Abell Andrew D; Wilce Matthew C J; Polyak Steven W^*; Booker Grant W

来源：Molecular Microbiology, 2014, 91(1): 110-120.

DOI：10.1111/mmi.12446

Protein biotinylation is catalysed by biotin protein ligase (BPL). The most characterized BPL is from Escherichia coli where it functions as both a biotin ligase and a homodimeric transcriptional repressor. Here we investigated another bifunctional BPL from the clinically important Staphylococcus aureus (SaBPL). Unliganded SaBPL (apo) exists in a dimer-monomer equilibrium at low micromolar concentrations - a stark contrast to E.coliBPL (EcBPL) that is monomeric under the same conditions. EMSA and SAXS analysis demonstrated that dimeric apo SaBPL adopted a conformation that was competent to bind DNA and necessary for it to function as a transcription factor. The SaBPL dimer-monomer dissociation constant was 5.8-fold tighter when binding the inhibitor biotin acetylene, but unchanged with biotin. F123, located in the dimer interface, was critical for homodimerization. Inhibition studies together with surface plasmon resonance analyses revealed a strong correlation between inhibitor potency and slow dissociation kinetics. A 24-fold difference in K-i values for these two enzymes was explained by differences in enzyme:inhibitor dissociation rates. Substitution of F123 in SaBPL and its equivalent in EcBPL altered both inhibitor potency and dissociation. Hence, F123 in SaBPL has novel roles in both protein dimerization and ligand-binding that have not been reported in EcBPL.

• 出版日期2014-1