Paillard, P. | bioRxiv Science

bioRxiv2026

Conjugative pilus tip structure reveals convergence in secretion systems

Bacteria deploy specialised secretion systems to transfer proteins and DNA into other cells, driving microbial evolution, virulence and the spread of antibiotic resistance1. Among these, type IV secretion systems (T4SSs) are unique in mediating both effector delivery and conjugative DNA transfer via extracellular pili that establish contact with recipient cells2. How these molecular machines couple recipient-cell engagement to the opening of a secretion channel is a central unresolved question across direct-contact secretion systems. Here we present the first high-resolution structure of a conjugative pilus tip from the R388 T4SS. The tip comprises the major pilin VirB2 and the minor pilin VirB5 together with two unexpected components: VirB6, repositioned from the inner-membrane stalk to the pilus apex where it forms a dynamic gate, and KikA, a modular distal adhesin likely involved in recipient-cell engagement. These findings unify pilus biogenesis, entry exclusion and recipient-cell engagement into a single structural mechanism in which VirB6 acts as a gatekeeper that couples tip contact to channel opening, while KikA shapes a pointed adhesin at the apex. By comparing this architecture to the type III and type VI secretion systems, we reveal convergent gating and penetration principles to contact-dependent bacterial secretion.

molecular biology↗

bioRxiv2026

HInt: interaction-based homology discovery through accelerated genome-scale AlphaFold screening

Identifying homologous proteins across deep evolutionary distances remains a major challenge because sequence and structural similarity progressively become undetectable over time. Although protein-protein interactions (PPIs) are often constrained by function and evolution, whether conserved interaction interfaces can provide an independent signal for homology detection has remained largely unexplored owing to the computational cost of proteome-scale interaction prediction. Here we introduce HInt (Homology by Interaction), an accelerated AlphaFold-based framework that enables practical proteome-scale PPI prediction through biologically informed pre-filtering and optimised high-throughput structure modelling. Using HInt, we establish interaction-based similarity as a third axis of homology detection. We show that conserved interaction interfaces reveal homologous relationships that remain inaccessible to conventional sequence- and structure-based approaches. Application of HInt to both prokaryotic and eukaryotic systems, together with experimental validation, uncovered a previously unrecognised VirB5 pilus-tip protein in the F-plasmid type IV secretion system and a previously unannotated F-box-like protein in the Saccharomyces cerevisiae ubiquitin-proteasome system. By enabling practical proteome-scale interaction screening, HInt provides a general framework for uncovering hidden homologues and expands the conceptual landscape of protein homology inference.

molecular biology↗