Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H(+):Cl(−) exchanger

Active exchangers dissipate the gradient of one substrate to accumulate nutrients, export xenobiotics and maintain cellular homeostasis. Mechanistic studies suggested that all exchangers share two fundamental properties: substrate binding is antagonistic and coupling is maintained by preventing shuttling of the empty transporter. The CLC Cl(−): H(+) exchangers control the homeostasis of cellular compartments in most living organisms but their transport mechanism remains unclear. We show that substrate binding to CLC-ec1 is synergistic rather than antagonistic: chloride binding induces protonation of a critical glutamate. The simultaneous binding of H(+) and Cl(−) gives rise to a fully-loaded state incompatible with conventional mechanisms. Mutations in the Cl(−) transport pathway identically alter the stoichiometries of Cl(−): H(+) exchange and binding. We propose that the thermodynamics of synergistic substrate binding determine the stoichiometry of transport rather than the kinetics of conformational changes and ion binding.