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

Abstract

Summary Protein biotinylation is catalysed by biotin protein ligase (BPL). The most characterized BPL is fromEscherichia 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 dimermonomer 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 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.

The Staphylococcus aureus group II biotin protein ligase BirA is an effective regulator of biotin operon transcription and requires the DNA binding domain for full enzymatic activity

“…SaBirA, SaBirA(btn), SaBirA(ATP) and SaBirA(Bio‐AMP) samples were prepared from the as purified protein as described in Experimental Procedures. These preparations were then assayed for DNA binding activity by EMSA using the buffer conditions given by Soares da Costa and coworkers (Soares da Costa et al ., ) (Fig. D).…”

“…These results parallel those seen in E. coli and B. subtilis upon overproduction of acceptor proteins (Cronan, ; Abdel‐Hamid and Cronan, ; Solbiati and Cronan, ; Chakravartty and Cronan, ; Henke and Cronan, ). Therefore, the SaBirA protein is a fully functional regulatory ligase which raised the question of how to explain the conflicting in vitro results reported by Soares da Costa and coworkers (Soares da Costa et al ., ).…”

“…D. SaBirA was incubated with either biotin (lanes 2–5) or ATP (lanes 7–10) and then treated with neutral hydroxylamine to remove any intermediate formed. Note the binding conditions were modified to those used by Soares da Costa and coworkers (Soares da Costa et al ., ) (the composition of the gels they utilized was not reported).…”

“…The putative S. aureus BirA contains an N‐terminal winged helix‐turn‐helix DNA binding domain. However, the protein was recently reported to dimerize in the absence of either biotin or Bio‐AMP with a Kd of 29 μM (Soares da Costa et al ., ), a concentration much lower than analogous preparations of E. coli BirA (Streaker et al ., ). Moreover, it was reported that S. aureus BirA bound the operator sequence in the absence of biotin and Bio‐AMP leading to a question of the biological function of the protein.…”

Structure and Function of Cyanobacterial DHDPS and DHDPR

“…Sedimentation velocity experiments were performed at 20 °C in a Beckman Coulter Model XL-A analytical ultracentrifuge using double sector quartz cells and An50-Ti rotor4950. 400 μl of buffer and 380 μl of sample at an initial concentration ranging from 0.1 μM to 7.0 μM were loaded into the reference and sample sectors of the cells, respectively.…”