A LARGE-SCALE EVOLUTIONARY AND STRUCTURAL ANALYSIS OF CLC CHANNELS AND TRANSPORTERS.

The CLC family of membrane transport proteins consists of chloride channels and anion/proton antiporters that share a similar structural scaffold. How the same fold accommodates two fundamentally distinct mechanisms is poorly understood, and while the current set of experimental structures provide some information, the changes appear limited. In this study, we show that it is possible to scale up the structural information available using AlphaFold2 predictions and identify additional structural differences associated with each mechanistic class. A phylogenetic analysis is carried out across all known CLC genes to expand the classification to include 569 channel and 1,051 transporter homologs that have been modeled. Using distance matrices, we validate AlphaFold2's ability to detect subtle structural differences among experimentally determined CLCs and use a random forest classifier to predict CLC channel vs. transporter sub-types to learn the structural changes that are the most important in the decision. The structural changes identified overlap with and contextualize the changes observed in experimental structures, expanding structural information across sequence space. The highest ranked change includes a contraction of distances between dimerization interface helices αH, αI, αP & αQ relative to the subunit core in the channel sub-types. This study lays out an approach for quantitative, large-scale structural analyses beyond experimental data and paves the way towards structural studies expanding on different conformational states and other protein families.