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.
Highlights
CLC transport proteins are a ubiquitous group of Cl− ion channels and Cl−/H+ antiporters that can be found in eukaryotes and bacteria
We have carried out SMD simulations to escort a Cl− ion through the pore of EcCLC to identify the accompanied local and global conformational changes of the protein
Umbrella sampling simulations at both the MM and QM/MM levels were performed to quantify the free-energy profile associated with Cl− transport
Summary
CLC transport proteins are a ubiquitous group of Cl− ion channels and Cl−/H+ antiporters that can be found in eukaryotes and bacteria. They are associated with many critical physiological and cellular processes such as aiding extreme acid-resistance response, cell-volume regulation, and muscle contraction (Maduke et al, 2000; Dutzler, 2006; Accardi and Picollo, 2010; Jentsch, 2015). Mutations in CLC proteins cause inherited diseases in humans, such as myotonia congenita, Dent’s disease, Bartter’s syndrome, osteopetrosis, and idiopathic epilepsy (Jentsch, 2008). CLC transport proteins assemble and function as homodimers, of which each monomer subunit contains an independent ion translocation pore (Chen, 2005). While CLC channels translocate Cl− ions passively, CLC antiporters mediate the coupled influx of Cl− and the efflux of H+ with a 2Cl−/1H+ ratio (Accardi and Miller, 2004; Picollo and Pusch, 2005; Scheel et al, 2005; Matulef and Maduke, 2007; Miller and Nguitragool, 2009; Feng et al, 2012; Accardi, 2015)
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