Centre for Protein Engineering, Macromolecules Biologiques Unit, University of Liège, Allée du 6 Août, 13 (B6A), Sart-Tilman, 4000 Liege, Belgium.
Biochem J. 2013 Mar 15;450(3):477-86. doi: 10.1042/BJ20121305.
MβL (metallo-β-lactamase) enzymes are usually produced by multi-resistant Gram-negative bacterial strains and have spread worldwide. An approach on the basis of phage display was used to select single-domain antibody fragments (VHHs, also called nanobodies) that would inhibit the clinically relevant VIM (Verona integron-encoded MβL)-4 MβL. Out of more than 50 selected nanobodies, only the NbVIM_38 nanobody inhibited VIM-4. The paratope, inhibition mechanism and epitope of the NbVIM_38 nanobody were then characterized. An alanine scan of the NbVIM_38 paratope showed that its binding was driven by hydrophobic amino acids. The inhibitory concentration was in the micromolar range for all β-lactams tested. In addition, the inhibition was found to follow a mixed hyperbolic profile with a predominantly uncompetitive component. Moreover, substrate inhibition was recorded only after nanobody binding. These kinetic data are indicative of a binding site that is distant from the active site. This finding was confirmed by epitope mapping analysis that was performed using peptides, and which identified two stretches of amino acids in the L6 loop and at the end of the α2 helix. Because this binding site is distant from the active site and alters both the substrate binding and catalytic properties of VIM-4, this nanobody can be considered as an allosteric inhibitor.
金属β-内酰胺酶（MβL）通常由多耐药革兰氏阴性菌产生，并已在全球范围内传播。本研究采用噬菌体展示技术筛选能够抑制临床相关 VIM（维罗纳整合子编码的 MβL）-4 MβL 的单域抗体片段（VHH，也称为纳米抗体）。在 50 多个筛选出的纳米抗体中，只有 NbVIM_38 纳米抗体抑制了 VIM-4。随后对 NbVIM_38 纳米抗体的抗原结合部位、抑制机制和表位进行了鉴定。NbVIM_38 抗原结合部位的丙氨酸扫描表明，其结合由疏水性氨基酸驱动。所有测试的β-内酰胺的抑制浓度均在微摩尔范围内。此外，发现抑制作用遵循混合双曲线特征，主要为非竞争性成分。此外，仅在纳米抗体结合后才记录到底物抑制。这些动力学数据表明存在一个远离活性部位的结合部位。通过使用肽进行的表位映射分析证实了这一发现，该分析确定了 L6 环和α2 螺旋末端的两个氨基酸延伸。由于该结合部位远离活性部位，并且改变了 VIM-4 的底物结合和催化特性，因此该纳米抗体可被视为变构抑制剂。
