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
School of Molecular and Biomedical Science, University of Adelaide, South Australia, 5005, Australia.
Mol Microbiol. 2014 Jan;91(1):110-20. doi: 10.1111/mmi.12446. Epub 2013 Nov 21.
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.
蛋白质生物素化由生物素蛋白连接酶（BPL）催化。最具特征的BPL来自大肠杆菌，它兼具生物素连接酶和同二聚体转录阻遏物的功能。在此，我们研究了来自具有临床重要性的金黄色葡萄球菌的另一种双功能BPL（SaBPL）。在低微摩尔浓度下，未结合配体的SaBPL（脱辅基蛋白）以二聚体 - 单体平衡形式存在，这与在相同条件下呈单体形式的大肠杆菌BPL（EcBPL）形成鲜明对比。电泳迁移率变动分析（EMSA）和小角X射线散射（SAXS）分析表明，二聚体脱辅基蛋白SaBPL采用了一种能够结合DNA的构象，这是其作为转录因子发挥功能所必需的。当结合抑制剂生物素乙炔时，SaBPL二聚体 - 单体解离常数紧密了5.8倍，但与生物素结合时不变。位于二聚体界面的F123对同二聚化至关重要。抑制研究以及表面等离子体共振分析表明抑制剂效力与缓慢解离动力学之间存在很强的相关性。这两种酶的抑制常数（Ki）值相差24倍，这可以通过酶与抑制剂解离速率的差异来解释。替换SaBPL中的F123及其在EcBPL中的等效物会改变抑制剂的效力和解离情况。因此，SaBPL中的F123在蛋白质二聚化和配体结合中具有EcBPL中未报道的新作用。
