A two-factor authentication mechanism licenses pilins for pilus assembly in gram-positive bacteria.
Gram-positive bacteria display virulence-associated pili that facilitate adhesion and biofilm formation. These pili are covalently polymerized by class C sortase enzymes, which selectively recognize their cognate pilin substrates amid numerous cell wall sorting signal (CWSS)-bearing proteins. The molecular basis for this stringent substrate specificity has remained unclear. Here, we develop a rapid, quantitative fluorescence-activated cell sorting assay to monitor pilus assembly in Corynebacterium diphtheriae, enabling high-throughput analysis of SpaA pilin and SrtA sortase variants. Using this platform, together with molecular modeling and dynamics simulations, we show that SrtA engages nearly the entire SpaA CWSS to form a membrane-embedded complex that incorporates not only the LPXTG motif but also its connector and transmembrane helix elements. Formation of this interface displaces an inhibitory active-site lid and activates the enzyme to load the pilin substrate. Systematic CWSS swapping experiments and deep mutational scanning further support this model, demonstrating that noncognate pilins are excluded because they fail to form the required interface. Conversely, SrtA variants with an artificially unlatched lid bypass the need for this interface, indicating that membrane-driven complex formation is important for substrate licensing. Together, these findings define a "two-factor authentication" mechanism for pilus assembly in gram-positive bacteria: class C sortases first verify pilin identity by forming a membrane-embedded interface that activates the enzyme, then they recognize the LPXTG motif to initiate loading and crosslinking. This work provides a unified molecular framework for selective pilin incorporation in gram-positive bacteria and identifies potential vulnerabilities in the licensing machinery that may be exploited therapeutically.