Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, Roque Sáenz Peña 325, 1876 Bernal, Buenos Aires, Argentina.
Protein Sci. 2012 Jul;21(7):964-76. doi: 10.1002/pro.2076. Epub 2012 May 31.
β-lactamases confer antibiotic resistance, one of the most serious world-wide health problems, and are an excellent theoretical and experimental model in the study of protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class-A β-lactamase with three tryptophan residues located in the protein core. Here, we report the 1.7-Å resolution X-ray structure, catalytic parameters, and thermodynamic stability of ESP(ΔW), an engineered mutant of ESP in which phenylalanine replaces the wild-type tryptophan residues. The structure revealed no qualitative conformational changes compared with thirteen previously reported structures of B. licheniformis β-lactamases (RMSD = 0.4-1.2 Å). However, a closer scrutiny showed that the mutations result in an overall more compact structure, with most atoms shifted toward the geometric center of the molecule. Thus, ESP(ΔW) has a significantly smaller radius of gyration (R(g)) than the other B. licheniformis β-lactamases characterized so far. Indeed, ESP(ΔW) has the smallest R(g) among 126 Class-A β-lactamases in the Protein Data Bank (PDB). Other measures of compactness, like the number of atoms in fixed volumes and the number and average of noncovalent distances, confirmed the effect. ESP(ΔW) proves that the compactness of the native state can be enhanced by protein engineering and establishes a new lower limit to the compactness of the Class-A β-lactamase fold. As the condensation achieved by the native state is a paramount notion in protein folding, this result may contribute to a better understanding of how the sequence determines the conformational variability and thermodynamic stability of a given fold.
β-内酰胺酶赋予抗生素耐药性，这是全球最严重的健康问题之一，也是研究蛋白质结构、动力学和进化的绝佳理论和实验模型。地衣芽孢杆菌外切小分子青霉素酶（ESP）是一种 A 类β-内酰胺酶，其蛋白质核心中有 3 个色氨酸残基。在这里，我们报道了 ESP（ΔW）的 1.7Å 分辨率 X 射线结构、催化参数和热力学稳定性，ESP（ΔW）是 ESP 的一种工程突变体，其中苯丙氨酸取代了野生型色氨酸残基。与之前报道的 13 种地衣芽孢杆菌β-内酰胺酶的结构（RMSD = 0.4-1.2Å）相比，该结构没有显示出定性的构象变化。然而，仔细观察表明，突变导致整体结构更加紧凑，大多数原子向分子的几何中心移动。因此，ESP（ΔW）的回转半径（R(g)）比迄今为止表征的其他地衣芽孢杆菌β-内酰胺酶小得多。事实上，ESP（ΔW）在蛋白质数据库（PDB）中 126 种 A 类β-内酰胺酶中具有最小的 R(g)。其他紧凑性指标，如固定体积中的原子数、非共价距离的数量和平均值，也证实了这一效果。ESP（ΔW）证明了通过蛋白质工程可以增强天然状态的紧凑性，并为 A 类β-内酰胺酶折叠的紧凑性建立了新的下限。由于天然状态下的凝聚是蛋白质折叠的一个重要概念，这一结果可能有助于更好地理解序列如何决定给定折叠的构象可变性和热力学稳定性。
