AbstractThe C-type lectin receptor langerin plays a vital role in the mammalian defense against invading pathogens. Its function hinges on the affinity to its co-factor Ca2+which in turn is regulated by the pH. We studied the structural consequences of protonating the allosteric pH-sensor histidine H294 by molecular dynamics simulations (total simulation time: about 120 µs) and Markov models. We discovered a mechanism in which the signal that the pH has dropped is transferred to the Ca2+-binding site without transferring the initial proton. Instead, protonation of H294 unlocks a conformation in which a protonated lysine side-chain forms a hydrogen bond with a Ca2+-coordinating aspartic acid. This destabilizes Ca2+in the binding pocket, which we probed by steered molecular dynamics. After Ca2+-release, the proton is likely transferred to the aspartic acid and stabilized by a dyad with a nearby glutamic acid, triggering a conformational transition and thus preventing Ca2+-rebinding.