Protein structure encodes unique information about the evolution of life on Earth. Yet structural data have been underutilised in evolutionary biology due to limited availability of high-quality models and analytical frameworks. Recent advances in computational structure prediction now enable large scale integration of structural, phylogenetic and functional data to explore molecular evolution in unprecedented detail.
We investigated the bacterial flagellar stator proteins MotA and MotB and relatives to understand how structural variation underpins evolutionary diversification. These proteins form ion channels that couple ion flux to torque generation, powering bacterial motility. Despite their key function, the evolutionary origin and structural diversity of stator complexes remain poorly understood.
We identified homologues across 27 bacterial phyla, reconstructed phylogenies, inferred ancestral sequences, and predicted structures for both extant and ancestral proteins using AlphaFold. Our analyses revealed two distinct groups: flagellar ion transporters (FIT) and generic ion transporters (GIT). FIT proteins share a conserved square-fold domain and a torque-generating interface, whereas GIT proteins are structurally heterogeneous and lack key motility-associated features. Despite this divergence, both groups retain conserved A–B subunit interactions and neighbouring gene organisation. Functional assays in Escherichia coli confirm that FIT specific structural elements are indispensable for motility.
Together, these findings suggest that the stator motor complex evolved once from an ancestral ion transporter and acquired specialised structural traits to enable torque generation. This integrative approach provides a powerful framework for investigating molecular evolution and diversity and highlights how protein structures capture the innovations that drive biological complexity.