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 have suggested that two fundamental properties are shared by all exchangers: substrate binding is antagonistic, and coupling is maintained by preventing shuttling of the empty transporter. The CLC H(+)/Cl(-) 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 crucial glutamate. The simultaneous binding of H(+) and Cl(-) gives rise to a fully loaded state that is incompatible with conventional transport mechanisms. Mutations in the Cl(-) transport pathway identically alter the stoichiometries of H(+)/Cl(-) exchange and binding. We propose that the thermodynamics of synergistic substrate binding, rather than the kinetics of conformational changes and ion binding, determine the stoichiometry of transport.