Tomatis Pablo E, Fabiane Stella M, Simona Fabio, Carloni Paolo, Sutton Brian J, Vila Alejandro J
Instituto de Biología Molecular y Celular de Rosario (IBR), Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina, Biophysics Section, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha.
Proc Natl Acad Sci U S A. 2008 Dec 30;105(52):20605-10. doi: 10.1073/pnas.0807989106. Epub 2008 Dec 19.
Protein evolution is crucial for organismal adaptation and fitness. This process takes place by shaping a given 3-dimensional fold for its particular biochemical function within the metabolic requirements and constraints of the environment. The complex interplay between sequence, structure, functionality, and stability that gives rise to a particular phenotype has limited the identification of traits acquired through evolution. This is further complicated by the fact that mutations are pleiotropic, and interactions between mutations are not always understood. Antibiotic resistance mediated by beta-lactamases represents an evolutionary paradigm in which organismal fitness depends on the catalytic efficiency of a single enzyme. Based on this, we have dissected the structural and mechanistic features acquired by an optimized metallo-beta-lactamase (MbetaL) obtained by directed evolution. We show that antibiotic resistance mediated by this enzyme is driven by 2 mutations with sign epistasis. One mutation stabilizes a catalytically relevant intermediate by fine tuning the position of 1 metal ion; whereas the other acts by augmenting the protein flexibility. We found that enzyme evolution (and the associated antibiotic resistance) occurred at the expense of the protein stability, revealing that MbetaLs have not exhausted their stability threshold. Our results demonstrate that flexibility is an essential trait that can be acquired during evolution on stable protein scaffolds. Directed evolution aided by a thorough characterization of the selected proteins can be successfully used to predict future evolutionary events and design inhibitors with an evolutionary perspective.
蛋白质进化对于生物体的适应和健康至关重要。这个过程通过在环境的代谢需求和限制范围内，为特定的生化功能塑造特定的三维折叠来实现。序列、结构、功能和稳定性之间复杂的相互作用产生了特定的表型，这限制了对通过进化获得的性状的识别。突变具有多效性，而且突变之间的相互作用并不总是被理解，这使得情况更加复杂。由β-内酰胺酶介导的抗生素耐药性代表了一种进化范式，其中生物体的适应性取决于单一酶的催化效率。基于此，我们剖析了通过定向进化获得的优化金属β-内酰胺酶（MbetaL）所获得的结构和机制特征。我们表明，这种酶介导的抗生素耐药性是由两个具有符号上位性的突变驱动的。一个突变通过微调一个金属离子的位置来稳定催化相关的中间体；而另一个突变则通过增加蛋白质的灵活性来起作用。我们发现酶的进化（以及相关的抗生素耐药性）是以蛋白质稳定性为代价发生的，这表明MbetaL尚未耗尽其稳定性阈值。我们的结果表明，灵活性是一种可以在稳定的蛋白质支架进化过程中获得的重要特性。通过对所选蛋白质进行全面表征辅助的定向进化可以成功地用于预测未来的进化事件，并从进化的角度设计抑制剂。
