Synergistic substrate binding determines the stoichiometry of transport of a prokaryotic H+/Cl− exchanger

Abstract

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.

Water access points and hydration pathways in CLC H + /Cl − transporters

“…Although much less is known about the H + translocation pathway (and mechanism), experimental studies have provided key information on the involvement of specific residues in H + transport (9,13,14,20,22,27,28). Extensive site-directed mutagenesis studies have zeroed in on two glutamate residues essential for H + transport (Fig.…”

“…Detailed structural and functional studies of 11,[18][19][20][21][22][23][24][25][26][27] have shed light on some of its key mechanistic aspects. Most prominently, these studies have characterized the Cl − permeation pathway and its lining residues (10,18,25) and established the role of E148, also known as Glu ex , as the extracellular gate for the Cl − pathway (9, 11).Although much less is known about the H + translocation pathway (and mechanism), experimental studies have provided key information on the involvement of specific residues in H + transport (9,13,14,20,22,27,28). Extensive site-directed mutagenesis studies have zeroed in on two glutamate residues essential for H + transport (Fig.…”

“…ClC-ec1, a CLC from Escherichia coli, has served as the prototype CLC for biophysical studies because of its known crystal structures (10, 11), its tractable biochemical behavior, and its structural and mechanistic similarities to mammalian CLC transporters (3)(4)(5)(6)(7)(8)(12)(13)(14)(15)(16)(17). Detailed structural and functional studies of 11,[18][19][20][21][22][23][24][25][26][27] have shed light on some of its key mechanistic aspects. Most prominently, these studies have characterized the Cl − permeation pathway and its lining residues (10,18,25) and established the role of E148, also known as Glu ex , as the extracellular gate for the Cl − pathway (9, 11).…”

“…Neutralization of either glutamate eliminates H + translocation by ClC-ec1 (9, 28). However, the discovery of these H + binding sites also raised a mechanistic puzzle (3, 23): How do protons translocate between the two sites, which are separated by a ∼15-Å-long, largely hydrophobic region within the lumen of the protein?Since the report of its first crystal structure, a large number of computational studies have aimed at investigating various molecular details related to the CLC H + transport mechanism (27,(29)(30)(31)(32)(33)(34). One model emerging from these studies proposes that water molecules may connect the two H + sites (Glu ex and Glu in ) and, thereby, facilitate H + transport (29,30,34).…”

“…Extensive site-directed mutagenesis studies have zeroed in on two glutamate residues essential for H + transport (Fig. 1A): E148 (Glu ex ), which acts as the main extracellular H + binding site (9,11,27), and E203 (Glu in ), which plays a similar role on the cytoplasmic side (20, 22, 28). Neutralization of either glutamate eliminates H + translocation by ClC-ec1 (9, 28).…”