Co‐evolution at protein–protein interfaces guides inference of stoichiometry of oligomeric protein complexes by de novo structure prediction

Abstract

The quaternary structure with specific stoichiometry is pivotal to the specific function of protein complexes. However, determining the structure of many protein complexes experimentally remains a major bottleneck. Structural bioinformatics approaches, such as the deep learning algorithm Alphafold2‐multimer (AF2‐multimer), leverage the co‐evolution of amino acids and sequence‐structure relationships for accurate de novo structure and contact prediction. Pseudo‐likelihood maximization direct coupling analysis (plmDCA) has been used to detect co‐evolving residue pairs by statistical modeling. Here, we provide evidence that combining both methods can be used for de novo prediction of the quaternary structure and stoichiometry of a protein complex. We achieve this by augmenting the existing AF2‐multimer confidence metrics with an interpretable score to identify the complex with an optimal fraction of native contacts of co‐evolving residue pairs at intermolecular interfaces. We use this strategy to predict the quaternary structure and non‐trivial stoichiometries of Bacillus subtilis spore germination protein complexes with unknown structures. Co‐evolution at intermolecular interfaces may therefore synergize with AI‐based de novo quaternary structure prediction of structurally uncharacterized bacterial protein complexes.

SpoVAF and FigP assemble into oligomeric ion channels that enhance spore germination

“…AlphaFold2-Multimer also predicts that GerAA and, separately, 5AF can form high-confidence hexamers ( Supplemental Fig. S1C ; Kilian and Bischofs 2023 ). For clarity, we present the pentamer model throughout this article but are currently unable to experimentally distinguish between the two models.…”

“…GerAA and GerAB are polytopic membrane proteins, and GerAC is a lipoprotein ( Moir and Cooper 2015 ). GerAA is predicted to oligomerize into a high-confidence pentamer or hexamer with a membrane channel at its oligomeric interface ( Gao et al 2023 ; Kilian and Bischofs 2023 ). Each GerAA protomer in the complex interacts with a GerAB subunit that is responsible for L-alanine recognition ( Artzi et al 2021 ; Gao et al 2023 ).…”