Chloride Ion Transport by the E. coli CLC Cl−/H+ Antiporter: A Combined Quantum-Mechanical and Molecular-Mechanical Study

Abstract

We performed steered molecular dynamics (SMD) and umbrella sampling simulations of Cl− ion migration through the transmembrane domain of a prototypical E. coli CLC Cl−/H+ antiporter by employing combined quantum-mechanical (QM) and molecular-mechanical (MM) calculations. The SMD simulations revealed interesting conformational changes of the protein. While no large-amplitude motions of the protein were observed during pore opening, the side chain rotation of the protonated external gating residue Glu148 was found to be critical for full access of the channel entrance by Cl−. Moving the anion into the external binding site (Sext) induced small-amplitude shifting of the protein backbone at the N-terminal end of helix F. As Cl− traveled through the pore, rigid-body swinging motions of helix R separated it from helix D. Helix R returned to its original position once Cl− exited the channel. Population analysis based on polarized wavefunction from QM/MM calculations discovered significant (up to 20%) charge loss for Cl− along the ion translocation pathway inside the pore. The delocalized charge was redistributed onto the pore residues, especially the functional groups containing π bonds (e.g., the Tyr445 side chain), while the charges of the H atoms coordinating Cl− changed almost negligibly. Potentials of mean force computed from umbrella sampling at the QM/MM and MM levels both displayed barriers at the same locations near the pore entrance and exit. However, the QM/MM PMF showed higher barriers (~10 kcal/mol) than the MM PMF (~2 kcal/mol). Binding energy calculations indicated that the interactions between Cl− and certain pore residues were overestimated by the semi-empirical PM3 Hamiltonian and underestimated by the CHARMM36 force fields, both of which were employed in the umbrella sampling simulations. In particular, CHARMM36 underestimated binding interactions for the functional groups containing π bonds, missing the stabilizations of the Cl− ion due to electron delocalization. The results suggested that it is important to explore these quantum effects for accurate descriptions of the Cl− transport.

Adaptive Partitioning QM/MM for Molecular Dynamics Simulations: 6. Proton Transport through a Biological Channel

“… − ,− Unlike conventional antiporters, where substrates take turns occupying a single pathway, H + and Cl – can simultaneously bind to EcCLC and their transport pathways overlap. , Intriguingly, mutation of a single residue, the external gating E148, can convert this antiporter to a Cl – channel. , The operating mechanism of EcCLC is very complicated, which is to a large extent due to the coupling of the Cl – transport and H + relay in opposite directions. Many experimental ,,,,,− and computational ,,,,,− efforts have been devoted toward elucidating the acting mechanisms of this transporter. For example, it has been suggested by experiments and by computations that the Cl – ion bound in the central binding site (S cen ) may be transiently protonated by the migrating H + .…”

Molecular Mechanism of Acetate Transport through the Acetate Channel SatP

“…The negative charge of the acetate may even delocalize to the aromatic rings lining up the pore, which may lead to a smaller radius of the acetate and facilitate its permeation. 21 Once free of this constriction site, the energy barrier can be gradually reduced, and the passage of the acetate becomes much smoother. Acetate Transport Mechanism.…”

Local conformational dynamics regulating transport properties of a Cl−/H+ antiporter

“…A) based on crystal structures as well as numerous mutagenesis experiments . These pathways intersect in the central cavity around the S cen site, which is the most stable binding site confirmed by isothermal titration calorimetry (ITC) experiments and computational calculations of the free energy profile . Three anion binding sites, S int , S cen , and S ext , were identified in the anion transport pathway, while E148 and E203 were recognized as important proton binding sites .…”