Structure of an Engineered β-Lactamase Maltose Binding Protein Fusion Protein: Insights into Heterotropic Allosteric Regulation

Engineering novel allostery into existing proteins is a challenging endeavor to obtain novel sensors, therapeutic proteins, or modulate metabolic and cellular processes. The RG13 protein achieves such allostery by inserting a circularly permuted TEM-1 β-lactamase gene into the maltose binding protein (MBP). RG13 is positively regulated by maltose yet is, serendipitously, inhibited by Zn2+ at low µM concentration. To probe the structure and allostery of RG13, we crystallized RG13 in the presence of mM Zn2+ concentration and determined its structure. The structure reveals that the MBP and TEM-1 domains are in close proximity connected via two linkers and a zinc ion bridging both domains. By bridging both TEM-1 and MBP, Zn2+ acts to “twist tie” the linkers thereby partially dislodging a linker between the two domains from its original catalytically productive position in TEM-1. This linker 1 contains residues normally part of the TEM-1 active site including the critical β3 and β4 strands important for activity. Mutagenesis of residues comprising the crystallographically observed Zn2+ site only slightly affected Zn2+ inhibition 2- to 4-fold. Combined with previous mutagenesis results we therefore hypothesize the presence of two or more inter-domain mutually exclusive inhibitory Zn2+ sites. Mutagenesis and molecular modeling of an intact TEM-1 domain near MBP within the RG13 framework indicated a close surface proximity of the two domains with maltose switching being critically dependent on MBP linker anchoring residues and linker length. Structural analysis indicated that the linker attachment sites on MBP are at a site that, upon maltose binding, harbors both the largest local Cα distance changes and displays surface curvature changes, from concave to relatively flat becoming thus less sterically intrusive. Maltose activation and zinc inhibition of RG13 are hypothesized to have opposite effects on productive relaxation of the TEM-1 β3 linker region via steric and/or linker juxtapositioning mechanisms.

将新型别构效应（allostery）工程化引入现有蛋白质，是获取新型传感器、治疗性蛋白或调控代谢与细胞过程的一项极具挑战性的工作。RG13蛋白通过将环状置换的TEM-1 β-内酰胺酶（TEM-1 β-lactamase）基因插入麦芽糖结合蛋白（MBP，maltose binding protein）中，实现了这类别构效应。RG13可被麦芽糖正向调控，且意外发现其在低微摩尔浓度下会被Zn²+抑制。为探究RG13的结构与别构机制，我们在毫摩尔级浓度的Zn²+存在下对RG13进行了结晶，并解析了其晶体结构。该结构显示，MBP结构域与TEM-1结构域紧密相邻，通过两个连接肽（linker）以及一个桥接两个结构域的锌离子相连。Zn²+通过桥接TEM-1与MBP两个结构域，起到"twist tie"的作用，从而将两个结构域间的一条连接肽从其在TEM-1中原本具有催化活性的位置部分剥离。该连接肽1包含原本属于TEM-1活性位点的氨基酸残基，其中包括对酶活性至关重要的β3链与β4链。对晶体学观测到的Zn²+结合位点中的氨基酸残基进行诱变，仅使Zn²+的抑制活性产生2至4倍的轻微变化。结合此前的诱变实验结果，我们据此推测存在两个或更多结构域间互斥的抑制性Zn²+结合位点。在RG13的结构框架内，对MBP附近完整TEM-1结构域进行诱变与分子建模的结果显示，两个结构域的表面紧密相邻；麦芽糖的开关效应关键依赖于MBP上连接肽的锚定残基以及连接肽的长度。结构分析显示，MBP上的连接肽附着位点处于这样一个区域：当结合麦芽糖时，该区域会同时出现最大的局部α碳（Cα）距离变化，并表现出表面曲率变化——从凹面变为相对平坦的曲面，从而降低空间位阻干扰。我们推测，麦芽糖对RG13的激活与锌离子对其的抑制，会通过空间位阻和/或连接肽并置机制，对TEM-1的β3连接肽区域的有效松弛产生相反的影响。