Program in Chemical Biology, University of Michigan, Ann Arbor, MI 48109.
Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109.
Proc Natl Acad Sci U S A. 2024 Jun 18;121(25):e2403273121. doi: 10.1073/pnas.2403273121. Epub 2024 Jun 12.
In secondary active transporters, a relatively limited set of protein folds have evolved diverse solute transport functions. Because of the conformational changes inherent to transport, altering substrate specificity typically involves remodeling the entire structural landscape, limiting our understanding of how novel substrate specificities evolve. In the current work, we examine a structurally minimalist family of model transport proteins, the small multidrug resistance (SMR) transporters, to understand the molecular basis for the emergence of a novel substrate specificity. We engineer a selective SMR protein to promiscuously export quaternary ammonium antiseptics, similar to the activity of a clade of multidrug exporters in this family. Using combinatorial mutagenesis and deep sequencing, we identify the necessary and sufficient molecular determinants of this engineered activity. Using X-ray crystallography, solid-supported membrane electrophysiology, binding assays, and a proteoliposome-based quaternary ammonium antiseptic transport assay that we developed, we dissect the mechanistic contributions of these residues to substrate polyspecificity. We find that substrate preference changes not through modification of the residues that directly interact with the substrate but through mutations peripheral to the binding pocket. Our work provides molecular insight into substrate promiscuity among the SMRs and can be applied to understand multidrug export and the evolution of novel transport functions more generally.
在继发性主动转运体中，相对有限的蛋白质折叠结构进化出了多样化的溶质转运功能。由于转运过程中固有的构象变化，改变底物特异性通常需要重塑整个结构景观，这限制了我们对新的底物特异性如何进化的理解。在目前的工作中，我们研究了一组结构极简的模型转运蛋白，即小多重耐药（SMR）转运体，以了解出现新底物特异性的分子基础。我们设计了一种选择性的 SMR 蛋白，使其能够随意输出季铵盐类防腐剂，类似于该家族中多药外排蛋白的一个分支的活性。我们使用组合诱变和深度测序，确定了这种工程化活性的必要和充分的分子决定因素。我们使用 X 射线晶体学、固态支持膜电生理学、结合测定和我们开发的基于蛋白脂质体的季铵盐防腐剂转运测定，剖析了这些残基对底物多特异性的作用机制。我们发现，底物偏好的改变不是通过与底物直接相互作用的残基的修饰，而是通过结合口袋周围的突变。我们的工作为 SMR 中的底物混杂提供了分子见解，并可用于更普遍地理解多药外排和新的转运功能的进化。
