Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
J Mol Biol. 2011 Mar 11;406(5):730-44. doi: 10.1016/j.jmb.2011.01.014. Epub 2011 Jan 13.
β-Lactamase inhibitory protein (BLIP) binds and inhibits a diverse collection of class A β-lactamases. Widespread resistance to β-lactam antibiotics currently limits the treatment strategies for Staphylococcus infections. The goals of this study were to determine the binding affinity of BLIP for Staphylococcus aureus PC1 β-lactamase and to identify mutants that alter binding affinity. The BLIP inhibition constant (K(i)) for PC1 β-lactamase was measured at 350 nM, and isothermal titration calorimetry experiments indicated a binding constant (K(d)) of 380 nM. Twenty-three residue positions in BLIP that contact β-lactamase were randomized, and phage display was used to sort the libraries for tight binders to immobilized PC1 β-lactamase. The BLIP(K74G) mutant was the dominant clone selected, and it was found to inhibit the PC1 β-lactamase with a K(i) of 42 nM, while calorimetry indicated a K(d) of 26 nM. Molecular modeling studies suggested that BLIP binds weakly to the PC1 β-lactamase due to the presence of alanine at position 104 of PC1. This position is occupied by glutamate in the TEM-1 enzyme, where it forms a salt bridge with the BLIP residue Lys74 that is important for the stability of the complex. This hypothesis was confirmed by showing that the PC1(A104E) enzyme binds BLIP with 15-fold greater affinity than wild-type PC1 β-lactamase. Kinetic measurements indicated similar association rates for all complexes with variation in affinity due to altered dissociation rate constants, suggesting that changes in short-range interactions are responsible for the altered binding properties of the mutants.
β-内酰胺酶抑制蛋白（BLIP）结合并抑制多种 A 类β-内酰胺酶。目前，β-内酰胺类抗生素的广泛耐药性限制了金黄色葡萄球菌感染的治疗策略。本研究的目的是确定 BLIP 与金黄色葡萄球菌 PC1 内酰胺酶的结合亲和力，并鉴定改变结合亲和力的突变体。测定了 BLIP 对 PC1 内酰胺酶的抑制常数（K(i)）为 350 nM，等温滴定量热法实验表明结合常数（K(d)）为 380 nM。BLIP 与β-内酰胺酶接触的 23 个残基位置被随机化，并用噬菌体展示技术对文库进行分拣，以获得与固定化 PC1 内酰胺酶紧密结合的紧密结合物。BLIP（K74G）突变体是选择的优势克隆，发现其对 PC1 内酰胺酶的抑制常数（K(i)）为 42 nM，而量热法表明 K(d)为 26 nM。分子建模研究表明，由于 PC1 中的 104 位为丙氨酸，BLIP 与 PC1 内酰胺酶的结合较弱。该位置在 TEM-1 酶中被谷氨酸占据，它与 BLIP 残基 Lys74 形成盐桥，这对于复合物的稳定性很重要。通过证明 PC1(A104E)酶与 BLIP 的结合亲和力比野生型 PC1 内酰胺酶高 15 倍，证实了这一假设。动力学测量表明，所有复合物的结合速率相似，亲和力的变化是由于解离速率常数的改变，这表明短程相互作用的变化是导致突变体结合特性改变的原因。
