| 1. |
- Coincon, Mathieu, et al.
(författare)
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Crystal structures reveal the molecular basis of ion translocation in sodium/proton antiporters
- 2016
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Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 23:3, s. 248-255
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Tidskriftsartikel (refereegranskat)abstract
- To fully understand the transport mechanism of Na+/H+ exchangers, it is necessary to clearly establish the global rearrangements required to facilitate ion translocation. Currently, two different transport models have been proposed. Some reports have suggested that structural isomerization is achieved through large elevator-like rearrangements similar to those seen in the structurally unrelated sodium-coupled glutamate-transporter homolog Glt(ph). Others have proposed that only small domain movements are required for ion exchange, and a conventional rocking-bundle model has been proposed instead. Here, to resolve these differences, we report atomic-resolution structures of the same Na+/H+ antiporter (NapA from Thermus thermophilus) in both outward- and inward-facing conformations. These data combined with cross-linking, molecular dynamics simulations and isothermal calorimetry suggest that Na+/H+ antiporters provide alternating access to the ion-binding site by using elevator-like structural transitions.
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| 2. |
- Lee, Chiara, et al.
(författare)
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A two-domain elevator mechanism for sodium/proton antiport
- 2013
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 501:7468, s. 573-577
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Tidskriftsartikel (refereegranskat)abstract
- Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis1. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets(2). The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli(1,3), for which both electron microscopy and crystal structures are available(4-6). 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(1,4). Likemany 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(7). The only reported NhaA crystal structure so far is of the low pH inactivated form(4). Here we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus, at 3 angstrom resolution, solved from crystals grown at pH7.8. 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(1,8,9) directly, a role supported hereby molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 20 degrees against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second(3), Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.
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| 3. |
- Lee, Chiara, et al.
(författare)
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Crystal structure of the sodium-proton antiporter NhaA dimer and new mechanistic insights
- 2014
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Ingår i: The Journal of General Physiology. - : Rockefeller University Press. - 0022-1295 .- 1540-7748. ; 144:6, s. 529-544
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Tidskriftsartikel (refereegranskat)abstract
- Sodium-proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium-proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium-proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pK(a) calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium-proton antiport in NhaA.
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| 4. |
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| 5. |
- Matsuoka, Rei, et al.
(författare)
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Structure, mechanism and lipid-mediated remodeling of the mammalian Na+/H+ exchanger NHA2
- 2022
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Ingår i: Nature Structural & Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 29:2, s. 108-120
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Tidskriftsartikel (refereegranskat)abstract
- The Na+/H+ exchanger SLC9B2, also known as NHA2, correlates with the long-sought-after Na+/Li+ exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite the functional importance of NHA2, structural information and the molecular basis for its ion-exchange mechanism have been lacking. Here we report the cryo-EM structures of bison NHA2 in detergent and in nanodiscs, at 3.0 and 3.5 Å resolution, respectively. The bison NHA2 structure, together with solid-state membrane-based electrophysiology, establishes the molecular basis for electroneutral ion exchange. NHA2 consists of 14 transmembrane (TM) segments, rather than the 13 TMs previously observed in mammalian Na+/H+ exchangers (NHEs) and related bacterial antiporters. The additional N-terminal helix in NHA2 forms a unique homodimer interface with a large intracellular gap between the protomers, which closes in the presence of phosphoinositol lipids. We propose that the additional N-terminal helix has evolved as a lipid-mediated remodeling switch for the regulation of NHA2 activity.
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| 6. |
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| 7. |
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| 8. |
- Winkelmann, Iven, 1989-, et al.
(författare)
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Crystal structure of the Na+/H+ antiporter NhaA at active pH reveals the mechanistic basis for pH sensing
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- The strict exchange of protons for sodium ions across cell membranes by Na+/H+ exchangers is a fundamental mechanism for cell homeostasis. At active pH, Na+/H+ exchange can be modelled as competition between H+ and Na+ to an ion-binding site, harbouring either one or two aspartic-acid residues. Nevertheless, extensive analysis on the model Na+/H+ antiporter NhaA from Escherichia coli, has shown that residues on the cytoplasmic surface, termed the pH sensor, shifts the pH at which NhaA becomes active. It was unclear how to incorporate the pH senor model into an alternating-access mechanism based on the NhaA structure at inactive pH 4. Here, we report the crystal structure of NhaA at active pH 6.5, and to an improved resolution of 2.2 angstrom. We show that at pH 6.5, residues in the pH sensor rearrange to form new salt-bridge interactions involving key histidine residues that widen the inward-facing cavity. What we now refer to as a pH gate, triggers a conformational change that enables water and Na+ to access the ion-binding site, as supported by molecular dynamics (MD) simulations. Our work highlights a unique, channel-like switch prior to substrate translocation in a secondary-active transporter.
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| 9. |
- Winkelmann, Iven, et al.
(författare)
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Structure and elevator mechanism of the mammalian sodium/proton exchanger NHE9
- 2020
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Ingår i: EMBO Journal. - : EMBO. - 0261-4189 .- 1460-2075. ; 39:24
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Tidskriftsartikel (refereegranskat)abstract
- Na+/H+ exchangers (NHEs) are ancient membrane-bound nanoma- chines that work to regulate intracellular pH, sodium levels and cell volume. NHE activities contribute to the control of the cell cycle, cell proliferation, cell migration and vesicle trafficking. NHE dysfunction has been linked to many diseases, and they are targets of pharma- ceutical drugs. Despite their fundamental importance to cell home- ostasis and human physiology, structural information for the mammalian NHEs was lacking. Here, we report the cryogenic elec- tron microscopy structure of NHE isoform 9 (SLC9A9) from Equus caballus at 3.2 Å resolution, an endosomal isoform highly expressed in the brain and associated with autism spectrum (ASD) and atten- tion deficit hyperactivity (ADHD) disorders. Despite low sequence identity, the NHE9 architecture and ion-binding site are remarkably most similar to distantly related bacterial Na+/H+ antiporters with 13 transmembrane segments. Collectively, we reveal the conserved architecture of the NHE ion-binding site, their elevator-like structural transitions, the functional implications of autism disease mutations and the role of phosphoinositide lipids to promote homodimerization that, together, have important physiological ramifications.
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