Multiscale Simulations Reveal Key Aspects of the Proton Transport Mechanism in the ClC-ec1 Antiporter

Lee, Sangyun; Swanson, Jessica M. J.; Voth, Gregory A.

doi:10.1016/j.bpj.2016.02.014

Cited by 59 publications

(146 citation statements)

References 75 publications

(122 reference statements)

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“…However, the calculated time scale for PT for both cases was significantly faster than the experimentally measured turnover rate for the overall PT process, indicating that PT from E203 to E148 can occur regardless of whether Cl – cen is present. 20 These findings raised the question of which PT step would be rate-determining and how PT could be coupled to Cl – transport, motivating the present study.…”

Section: Introductionmentioning

confidence: 90%

“…27 Initial configurations for the simulations were obtained from a previous study of this system. 20 The error bars on the PMF calculations were estimated using block averaging by dividing each trajectory into four consecutive blocks.…”

Section: Methodsmentioning

confidence: 99%

“…We employed the MS-RMD model for E203 from our previous work 20 as residue 148 is approximately ∼15 Å from E203 and thus does not significantly change the electrostatic environment that primarily determines its MS-RMD parameters. A148 was treated by the CHARMM classical force field.…”

Section: Methodsmentioning

confidence: 99%

“…The PT rate constants were estimated using transition state theory as follows, 20,35 where k B is Boltzmann’s constant, T is the simulation temperature (300 K), and Δ F ⧧ is the free energy barrier height in the PMF. The fundamental frequency ω 0 is that of the reactant state oscillations around its minimum, which is defined aswhere r 0 is the local minimum in the PMFs.…”

Section: Methodsmentioning

confidence: 99%

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The Origin of Coupled Chloride and Proton Transport in a Cl^–/H⁺ Antiporter

et al. 2016

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The ClC family of transmembrane proteins functions throughout nature to control the transport of Cl– ions across biological membranes. ClC-ec1 from Escherichia coli is an antiporter, coupling the transport of Cl– and H+ ions in opposite directions and driven by the concentration gradients of the ions. Despite keen interest in this protein, the molecular mechanism of the Cl–/H+ coupling has not been fully elucidated. Here, we have used multiscale simulation to help identify the essential mechanism of the Cl–/H+ coupling. We find that the highest barrier for proton transport (PT) from the intra- to extracellular solution is attributable to a chemical reaction, the deprotonation of glutamic acid 148 (E148). This barrier is significantly reduced by the binding of Cl– in the “central” site (Cl–cen), which displaces E148 and thereby facilitates its deprotonation. Conversely, in the absence of Cl–cen E148 favors the “down” conformation, which results in a much higher cumulative rotation and deprotonation barrier that effectively blocks PT to the extracellular solution. Thus, the rotation of E148 plays a critical role in defining the Cl–/H+ coupling. As a control, we have also simulated PT in the ClC-ec1 E148A mutant to further understand the role of this residue. Replacement with a non-protonatable residue greatly increases the free energy barrier for PT from E203 to the extracellular solution, explaining the experimental result that PT in E148A is blocked whether or not Cl–cen is present. The results presented here suggest both how a chemical reaction can control the rate of PT and also how it can provide a mechanism for a coupling of the two ion transport processes.

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Section: Introductionmentioning

confidence: 90%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The Origin of Coupled Chloride and Proton Transport in a Cl^–/H⁺ Antiporter

et al. 2016

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“…To overcome this challenge, a multiscale reactive molecular dynamics (MS-RMD) method has been extensively developed and applied in our group to study the explicit PT process in aqueous and biological contexts, including CcO (e.g., refs. [23][24][25][26][27][28][29][30][31].…”

Section: Significancementioning

confidence: 99%

Understanding the essential proton-pumping kinetic gates and decoupling mutations in cytochrome c oxidase

Liang

Swanson

Wikström

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Cytochrome c oxidase (CcO) catalyzes the reduction of oxygen to water and uses the released free energy to pump protons against the transmembrane proton gradient. To better understand the proton-pumping mechanism of the wild-type (WT) CcO, much attention has been given to the mutation of amino acid residues along the proton translocating D-channel that impair, and sometimes decouple, proton pumping from the chemical catalysis.Although their influence has been clearly demonstrated experimentally, the underlying molecular mechanisms of these mutants remain unknown. In this work, we report multiscale reactive molecular dynamics simulations that characterize the free-energy profiles of explicit proton transport through several important D-channel mutants. Our results elucidate the mechanisms by which proton pumping is impaired, thus revealing key kinetic gating features in CcO. In the N139T and N139C mutants, proton back leakage through the D-channel is kinetically favored over proton pumping due to the loss of a kinetic gate in the N139 region. In the N139L mutant, the bulky L139 side chain inhibits timely reprotonation of E286 through the D-channel, which impairs both proton pumping and the chemical reaction. In the S200V/S201V double mutant, the proton affinity of E286 is increased, which slows down both proton pumping and the chemical catalysis. This work thus not only provides insight into the decoupling mechanisms of CcO mutants, but also explains how kinetic gating in the D-channel is imperative to achieving high proton-pumping efficiency in the WT CcO.proton pump | decoupling mutants | cytochrome c oxidase | proton transport | multiscale C ytochrome c oxidase (CcO) is the terminal enzyme of the respiratory electron transfer chain in the inner membrane of mitochondria and the plasma membrane of bacteria. It catalyzes the oxidation of cytochrome c molecules and reduction of O 2 to H 2 O. For the aa 3 -type CcO, found in mitochondria and many bacteria, the free energy available from the chemical reaction is used to pump one proton across the membrane per electron transferred to O 2 , generating the transmembrane electrochemical proton gradient necessary for ATP synthesis (1-3). Thus, four "substrate" protons are taken up from the mitochondrial matrix or bacterial cytoplasmic side (negatively charged; N-side) of the membrane and consumed in the reduction of O 2 , whereas another four "pumped" protons are transported to the intermembrane (or extracellular in bacteria) space (positively charged; P-side) of the membrane (Fig. 1). All four pumped protons and at least two of the substrate protons are taken up from solution on the N-side of the membrane by the D-channel, which begins with amino acid residue D132 and ends at residue E286 two-thirds of the way into the protein (4, 5). The remaining substrate protons are taken up by the K-channel (not shown in Fig. 1, but extensively discussed in ref. 6). Next, the protons are either transferred to the binuclear center (BNC) to react with O 2 or to a pump loading site (P...

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Diversity of functional alterations of the ClC‐5 exchanger in the region of the proton glutamate in patients with Dent disease 1

et al. 2021

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Mutations in the CLCN5 gene encoding the 2Cl − /1H + exchanger ClC-5 are associated with Dent disease 1, an inherited renal disorder characterized by low-molecular-weight (LMW) proteinuria and hypercalciuria. In the kidney, ClC-5 is mostly localized in proximal tubule cells, where it is thought to play a key role in the endocytosis of LMW proteins. Here, we investigated the consequences of eight previously reported pathogenic missense mutations of ClC-5 surrounding the "proton glutamate" that serves as a crucial H + -binding site for the exchanger. A complete loss of function was observed for a group of mutants that were either retained in the endoplasmic reticulum of HEK293T cells or unstainable at plasma membrane due to proteasomal degradation. In contrast, the currents measured for the second group of mutations in Xenopus laevis oocytes were reduced. Molecular dynamics simulations performed on a ClC-5 homology model demonstrated that such mutations might alter ClC-5 protonation by interfering with the water pathway. Analysis of clinical data from patients harboring these mutations demonstrated no phenotype/genotype correlation. This study reveals that mutations clustered in a crucial region of ClC-5 have diverse molecular consequences in patients with Dent disease 1, ranging from altered expression to defects in transport.

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Multiscale Simulations Reveal Key Aspects of the Proton Transport Mechanism in the ClC-ec1 Antiporter

Cited by 59 publications

References 75 publications

The Origin of Coupled Chloride and Proton Transport in a Cl^–/H⁺ Antiporter

The Origin of Coupled Chloride and Proton Transport in a Cl^–/H⁺ Antiporter

Understanding the essential proton-pumping kinetic gates and decoupling mutations in cytochrome c oxidase

Diversity of functional alterations of the ClC‐5 exchanger in the region of the proton glutamate in patients with Dent disease 1

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Multiscale Simulations Reveal Key Aspects of the Proton Transport Mechanism in the ClC-ec1 Antiporter

Cited by 59 publications

References 75 publications

The Origin of Coupled Chloride and Proton Transport in a Cl–/H+ Antiporter

The Origin of Coupled Chloride and Proton Transport in a Cl–/H+ Antiporter

Understanding the essential proton-pumping kinetic gates and decoupling mutations in cytochrome c oxidase

Diversity of functional alterations of the ClC‐5 exchanger in the region of the proton glutamate in patients with Dent disease 1

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The Origin of Coupled Chloride and Proton Transport in a Cl^–/H⁺ Antiporter

The Origin of Coupled Chloride and Proton Transport in a Cl^–/H⁺ Antiporter