Sequence variation among homologous proteins can shed light on their function and ancestry. In this study, we analyze variation at catalytic residues among MCR (mobile colistin resistance) proteins, which confer resistance to the last resort antibiotic, colistin, in gram-negative bacteria. We show that not all naturally occurring variants at a lipid A-binding residue, Ser284, are tolerated in MCR-1. In particular, the substitution of Ser284 with Asp, found naturally in MCR-5, resulted in diminished colistin resistance. Using phylogenetic analyses and structure predictions we trace back variation at this site among MCRs to their ancestors, i.e. EptA phosphoethanolamine transferases that are encoded by diverse bacterial genomes. Mutational studies and AlphaFold-based structural modeling revealed that the functional importance of position 284 varies between phylogenetically distant MCRs, i.e. MCR-1 and MCR-5. Despite a high degree of similarity among their catalytic domains, inter-domain interactions were not conserved between MCR-1 and MCR-5 due to their different ancestries, providing a mechanistic basis behind the different phenotypes of similar mutations at position 284. Our study thus uncovers subtle differences in the organization of domains among MCR proteins that can lead to substantial differences in their catalytic properties and mutational tolerances.

中文翻译：

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移动粘菌素抗性蛋白活性位点的序列变异通过结构域间相互作用在进化上被容纳。


同源蛋白之间的序列变异可以揭示它们的功能和祖先。在这项研究中，我们分析了 MCR（移动粘菌素抗性）蛋白之间催化残基的变异，这些蛋白在革兰氏阴性菌中赋予对最后手段抗生素粘菌素的耐药性。我们表明，并非所有脂质 A 结合残基 Ser284 处天然存在的变体都可以在 MCR-1 中耐受。特别是，在 MCR-5 中天然发现的 Ser284 被 Asp 取代，导致粘菌素耐药性降低。使用系统发育分析和结构预测，我们将 MCR 在该位点的变异追溯到它们的祖先，即由不同细菌基因组编码的 EptA 磷酸乙醇胺转移酶。突变研究和基于 AlphaFold 的结构建模表明，位置 284 的功能重要性在系统发育距离较远的 MCR 之间有所不同，即 MCR-1 和 MCR-5。尽管它们的催化结构域之间高度相似，但由于它们的祖先不同，MCR-1 和 MCR-5 之间的结构域间相互作用并不保守，这为在 284 位相似突变的不同表型提供了机制基础。因此，我们的研究揭示了 MCR 蛋白之间结构域组织的细微差异，这可能导致它们的催化特性和突变耐受性存在巨大差异。