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Dual roles of F 123 in protein homodimerization and inhibitor binding to biotin protein ligase from S taphylococcus aureus
Author(s) -
Soares da Costa Tatiana P.,
Yap Min Y.,
Perugini Matthew A.,
Wallace John C.,
Abell Andrew D.,
Wilce Matthew C. J.,
Polyak Steven W.,
Booker Grant W.
Publication year - 2014
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/mmi.12446
Subject(s) - dna ligase , biotin , dimer , dissociation constant , biotinylation , biochemistry , biology , ubiquitin ligase , plasma protein binding , monomer , enzyme , stereochemistry , chemistry , ubiquitin , receptor , organic chemistry , gene , polymer
Summary Protein biotinylation is catalysed by biotin protein ligase ( BPL ). The most characterized BPL is from E scherichia coli where it functions as both a biotin ligase and a homodimeric transcriptional repressor. Here we investigated another bifunctional BPL from the clinically important S taphylococcus aureus ( Sa BPL ). Unliganded Sa BPL (apo) exists in a dimer‐monomer equilibrium at low micromolar concentrations – a stark contrast to E . coli BPL ( Ec BPL ) that is monomeric under the same conditions. EMSA and SAXS analysis demonstrated that dimeric apo Sa BPL adopted a conformation that was competent to bind DNA and necessary for it to function as a transcription factor. The Sa BPL dimer‐monomer dissociation constant was 5.8‐fold tighter when binding the inhibitor biotin acetylene, but unchanged with biotin. F 123, 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 F 123 in Sa BPL and its equivalent in Ec BPL altered both inhibitor potency and dissociation. Hence, F 123 in Sa BPL has novel roles in both protein dimerization and ligand‐binding that have not been reported in Ec BPL .