title: Dual roles of F123 in protein homodimerization and inhibitor binding to biotin protein ligase from Staphylococcus aureus.
abstract: 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. coli BPL (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 Ki 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.
authors.affiliation: School of Molecular and Biomedical Science, University of Adelaide, South Australia, 5005, Australia.
authors.forename: Tatiana P
authors.initials: TP
authors.lastname: Soares da Costa
authors.forename: Min Y
authors.initials: MY
authors.lastname: Yap
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Perugini
authors.forename: John C
authors.initials: JC
authors.lastname: Wallace
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Abell
authors.forename: Matthew C J
authors.initials: MC
authors.lastname: Wilce
authors.forename: Steven W
authors.initials: SW
authors.lastname: Polyak
authors.forename: Grant W
authors.initials: GW
authors.lastname: Booker
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics / Q000502:physiology*; D001710:Biotin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D010649:Phenylalanine / Q000378:metabolism*; D011487:Protein Conformation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism; D053838:Scattering, Small Angle; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics; D020349:Surface Plasmon Resonance; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008024:Ligands; D012097:Repressor Proteins; D010649:Phenylalanine; D001710:Biotin; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli