Pathway-Dependent Energetics of SLC15A4–TASL Engagement: Single-Protomer Switching and Dimer-Interface Remodeling

Abstract Membrane-embedded signalosome assembly is co-gated by conformational selection, oligomeric interfaces, and the lipid microenvironment, yet short-lived, tightly coupled intermediate states are hard to infer from static structures. SLC15A4 recruits TASL to support IRF5 signaling, but available structures largely capture distinct conformational endpoints, leaving assembly bottlenecks, interface/lipid coupling, and inhibitor interception without a quantitative energetic rationale. Here, we developed a computational energetic framework connecting the resting apo dimer to a TASL-permissive state. In the apo state, SLC15A4 maintained a persistent dimeric arrangement organized around a TM9/TM12-centered interfacial scaffold, while the interfacial cholesterol molecules maintained persistent contacts with the interface; luminal hydrophobic packing and a multivalent hydrogen-bond network around N428 were associated with interface persistence, while the dimer retained characteristic coupled motions of MFS helical bundles. Along the prescribed insertion pathway, TASL exhibited a pronounced apparent free-energy rise near the pocket, accompanied by rapid contact formation and a single-protomer switch from luminal-open to cytosolic-open, yielding a modeled asymmetric intermediate with altered cross-protomer interface geometry. TASL was stabilized by directional anchoring at a few key cationic sites. Pathway-dependent free-energy profiles further indicated that TASL engagement relaxed luminal-side interfacial stabilization and lowered the free-energy cost of inter-subunit separation without compromising pocket binding, potentially contributing to the formation of a signaling-competent SLC15A4–TASL module. Finally, our results suggest that an inhibitor limits access to the TASL-permissive state by locking the luminal-open conformation and occupying the central cavity, increasing the free-energy cost along the prescribed TASL entry and reshaping electrostatic determinants. Together, these results connect experimentally resolved structural states through a computational energetic framework in which TASL-associated single-protomer switching may couple to luminal interface weakening and reduced interface persistence, motivating therapeutic strategies targeting the SLC15A4–TASL–IRF5 axis via conformational locking and competitive cavity occupation.