Q-cubed mutant cues clues to CLC antiport mechanism.
Ontology highlight
ABSTRACT:
SUBMITTER: Miller C
PROVIDER: S-EPMC7894039 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:ClC chloride channels and transporters play major roles in cellular excitability, epithelial salt transport, volume, pH, and blood pressure regulation. One family member, ClC-ec1 from Escherichia coli, has been structurally resolved crystallographically and subjected to intensive mutagenetic, crystallographic, and electrophysiological studies. It functions as a Cl(-)/H(+) antiporter, not a Cl(-) channel; however, the molecular mechanism for Cl(-)/H(+) exchange is largely unknown. Using all-atom normal-mode analysis to explore possible mechanisms for this antiport, we propose that Cl(-)/H(+) exchange involves a conformational cycle of alternating exposure of Cl(-) and H(+) binding sites of both ClC pores to the two sides of the membrane. Both pores switch simultaneously from facing outward to facing inward, reminiscent of the standard alternating-access mechanism, which may have direct implications for eukaryotic Cl(-)/H(+) transporters and Cl(-) channels.
| S-EPMC2849085 | biostudies-literature
Project description:Among coupled exchangers, CLCs uniquely catalyze the exchange of oppositely charged ions (Cl- for H+). Transport-cycle models to describe and explain this unusual mechanism have been proposed based on known CLC structures. While the proposed models harmonize with many experimental findings, gaps and inconsistencies in our understanding have remained. One limitation has been that global conformational change - which occurs in all conventional transporter mechanisms - has not been observed in any high-resolution structure. Here, we describe the 2.6 Å structure of a CLC mutant designed to mimic the fully H+-loaded transporter. This structure reveals a global conformational change to improve accessibility for the Cl- substrate from the extracellular side and new conformations for two key glutamate residues. Together with DEER measurements, MD simulations, and functional studies, this new structure provides evidence for a unified model of H+/Cl- transport that reconciles existing data on all CLC-type proteins.
| S-EPMC7253180 | biostudies-literature
Project description:Sodium/proton (Na(+)/H(+)) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets. The best understood model system for Na(+)/H(+) antiport is NhaA from Escherichia coli, for which both electron microscopy and crystal structures are available. NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein. Like many Na(+)/H(+) antiporters, the activity of NhaA is regulated by pH, only becoming active above pH?6.5, at which point a conformational change is thought to occur. The only reported NhaA crystal structure so far is of the low pH inactivated form. Here we describe the active-state structure of a Na(+)/H(+) antiporter, NapA from Thermus thermophilus, at 3?Å resolution, solved from crystals grown at pH?7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 20° against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500?ions?per second, Na(+)/H(+) antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.
| S-EPMC3914025 | biostudies-literature
Project description:ClC chloride channels are voltage-gated transmembrane proteins that have been associated with a wide range of regulatory roles in vertebrates. To accomplish their function, they allow small inorganic anions to efficiently pass through, while blocking the passage of all other particles. Understanding the conduction mechanism of ClC has been the subject of many experimental investigations, but until now, the detailed dynamic mechanism was not known despite the availability of crystallographic structures. We investigate Cl(-) conduction by means of an all-atom molecular dynamics simulation of the ClC channel in a membrane environment. Based on our simulation results, we propose a king-of-the-hill mechanism for permeation, in which a lone ion bound to the center of the ClC pore is pushed out by a second ion that enters the pore and takes its place. Although the energy required to extract the single central ion from the pore is enormous, by resorting to this two-ion process, the largest free energy barrier for conduction is reduced to 4 kcal/mol. At the narrowest part of the pore, residues Tyr-445 and Ser-107 stabilize the central ion. There, the bound ion blocks the pore, disrupting the formation of a continuous water file that could leak protons, possibly preventing the passage of uncharged solutes.
| S-EPMC1303931 | biostudies-literature
Project description:NarK belongs to the nitrate/nitrite porter (NNP) family in the major facilitator superfamily (MFS) and plays a central role in nitrate uptake across the membrane in diverse organisms, including archaea, bacteria, fungi and plants. Although previous studies provided insight into the overall structure and the substrate recognition of NarK, its molecular mechanism, including the driving force for nitrate transport, remained elusive. Here we demonstrate that NarK is a nitrate/nitrite antiporter, using an in vitro reconstituted system. Furthermore, we present the high-resolution crystal structures of NarK from Escherichia coli in the nitrate-bound occluded, nitrate-bound inward-open and apo inward-open states. The integrated structural, functional and computational analyses reveal the nitrate/nitrite antiport mechanism of NarK, in which substrate recognition is coupled to the transport cycle by the concomitant movement of the transmembrane helices and the key tyrosine and arginine residues in the substrate-binding site.
| S-EPMC4432589 | biostudies-literature
Project description:LAT1 (SLC7A5) is one of the most studied membrane transporters due to its relevance to physiology in supplying essential amino acids to brain and fetus, and to pathology being linked to nervous or embryo alterations; moreover, LAT1 over-expression is always associated with cancer development. Thus, LAT1 is exploited as a pro-drug vehicle and as a target for anti-cancer therapy. We here report the identification of a new substrate with pathophysiological implications, i.e., Cu-histidinate, and an unconventional uniport mechanism exploited for the Cu-histidinate transport. Crystals of the monomeric species Cu(His)2 were obtained in our experimental conditions and the actual transport of the complex was evaluated by a combined strategy of bioinformatics, site-directed mutagenesis, radiolabeled transport, and mass spectrometry analysis. The LAT1-mediated transport of Cu(His)2 may have profound implications for both the treatment of copper dysmetabolism diseases, such as the rare Menkes disease, and of cancer as an alternative to platinum-based therapies.
| S-EPMC10492218 | biostudies-literature
Project description:ClC-0 is a chloride channel whose gating is sensitive to both voltage and chloride. Based on analysis of gating kinetics using single-channel recordings, a five-state model was proposed to describe the dependence of ClC-0 fast-gate opening on voltage and external chloride (Chen, T.-Y., and C. Miller. 1996. J. Gen. Physiol. 108:237-250). We aimed to use this five-state model as a starting point for understanding the structural changes that occur during gating. Using macroscopic patch recordings, we were able to reproduce the effects of voltage and chloride that were reported by Chen and Miller and to fit our opening rate constant data to the five-state model. Upon further analysis of both our data and those of Chen and Miller, we learned that in contrast to their conclusions, (a) the features in the data are not adequate to rule out a simpler four-state model, and (b) the chloride-binding step is voltage dependent. In order to be able to evaluate the effects of mutants on gating (described in the companion paper, see Engh et al. on p. 351 of this issue), we developed a method for determining the error on gating model parameters, and evaluated the sources of this error. To begin to mesh the kinetic model(s) with the known CLC structures, a model of ClC-0 was generated computationally based on the X-ray crystal structure of the prokaryotic homolog ClC-ec1. Analysis of pore electrostatics in this homology model suggests that at least two of the conclusions derived from the gating kinetics analysis are consistent with the known CLC structures: (1) chloride binding is necessary for channel opening, and (2) chloride binding to any of the three known chloride-binding sites must be voltage dependent.
| S-EPMC2151655 | biostudies-literature
Project description:Mutations in the transcription factor p53 are among the most common genetic alterations in human cancer, and missense p53 mutations in cancer cells can lead to aggressive phenotypes. So far, only few studies investigated transcriptional reprogramming under mutant p53 expression as a means to identify deregulated targets and pathways. A review of the literature was carried out focusing on mutant p53-dependent transcriptome changes with the aims of (i) verifying whether different p53 mutations can be equivalent for their effects, or whether there is a mutation-specific transcriptional reprogramming of target genes, (ii) understanding what is the main mechanism at the basis of upregulation or downregulation of gene expression under the p53 mutant background, (iii) identifying novel candidate target genes of WT and/or mutant p53 and (iv) defining cellular pathways affected by the mutant p53-dependent gene expression reprogramming. Nearly 600 genes were consistently found upregulated or downregulated upon ectopic expression of mutant p53, regardless of the specific p53 mutation studied. Promoter analysis and the use of ChIP-seq data indicate that, for most genes, the expression changes could be ascribed to a loss both of WT p53 transcriptional activation and repressor functions. Pathway analysis indicated changes in the metabolism/catabolism of amino acids such as aspartate, glutamate, arginine and proline. Novel p53 candidate target genes were also identified, including ARID3B, ARNT2, CLMN, FADS1, FTH1, KPNA2, LPHN2, PARD6B, PDE4C, PIAS2, PRPF40A, PYGL and RHOBTB2, involved in the metabolism, xenobiotic responses and cell differentiation.
| S-EPMC3740285 | biostudies-literature
Project description:Unveiling Errors in Soil Microbial Community Sequencing: A Case for Reference Soils and Improved Diagnostics for Nanopore Sequencing
| PRJEB75585 | ENA