Verna and Marrs McLean Department of Biochemistry and Molecular Pharmacology, Baylor College of Medicine, Houston, TX 77030.
Department of Molecular Biophysics and Integrated Bioimaging, Berkeley Center for Structural Biology Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
Proc Natl Acad Sci U S A. 2024 Mar 19;121(12):e2313513121. doi: 10.1073/pnas.2313513121. Epub 2024 Mar 14.
Cooperative interactions between amino acids are critical for protein function. A genetic reflection of cooperativity is epistasis, which is when a change in the amino acid at one position changes the sequence requirements at another position. To assess epistasis within an enzyme active site, we utilized CTX-M β-lactamase as a model system. CTX-M hydrolyzes β-lactam antibiotics to provide antibiotic resistance, allowing a simple functional selection for rapid sorting of modified enzymes. We created all pairwise mutations across 17 active site positions in the β-lactamase enzyme and quantitated the function of variants against two β-lactam antibiotics using next-generation sequencing. Context-dependent sequence requirements were determined by comparing the antibiotic resistance function of double mutations across the CTX-M active site to their predicted function based on the constituent single mutations, revealing both positive epistasis (synergistic interactions) and negative epistasis (antagonistic interactions) between amino acid substitutions. The resulting trends demonstrate that positive epistasis is present throughout the active site, that epistasis between residues is mediated through substrate interactions, and that residues more tolerant to substitutions serve as generic compensators which are responsible for many cases of positive epistasis. Additionally, we show that a key catalytic residue (Glu166) is amenable to compensatory mutations, and we characterize one such double mutant (E166Y/N170G) that acts by an altered catalytic mechanism. These findings shed light on the unique biochemical factors that drive epistasis within an enzyme active site and will inform enzyme engineering efforts by bridging the gap between amino acid sequence and catalytic function.
氨基酸之间的合作相互作用对于蛋白质功能至关重要。合作的遗传反映是上位性，即一个位置的氨基酸变化改变了另一个位置的序列要求。为了评估酶活性部位内的上位性，我们利用 CTX-M β-内酰胺酶作为模型系统。CTX-M 水解β-内酰胺抗生素以提供抗生素耐药性，从而可以通过简单的功能选择快速对修饰酶进行分类。我们在β-内酰胺酶酶的活性部位的 17 个位置上创建了所有的成对突变，并使用下一代测序技术对两种β-内酰胺抗生素的变体功能进行定量。通过比较 CTX-M 活性部位中双突变的抗生素耐药性功能与其基于组成单突变的预测功能，确定了上下文相关的序列要求，从而揭示了氨基酸取代之间的正上位性（协同相互作用）和负上位性（拮抗相互作用）。由此产生的趋势表明，正上位性存在于整个活性部位，残基之间的上位性通过底物相互作用介导，并且对取代更耐受的残基充当通用补偿者，这是许多正上位性的原因。此外，我们表明关键催化残基（Glu166）可发生补偿性突变，并对一种这样的双突变体（E166Y/N170G）进行了表征，该突变体通过改变的催化机制起作用。这些发现揭示了驱动酶活性部位内上位性的独特生化因素，并通过在氨基酸序列和催化功能之间架起桥梁，为酶工程努力提供信息。
