The Fourth Transmembrane Domain of the Helicobacter pylori Na+/H+ Antiporter NhaA Faces a Water-filled Channel Required for Ion Transport

Kuwabara, N.; Inoue, Hiroki; Tsuboi, Yoshikatsu; Nakamura, Norihiro; Kanazawa, Hiroshi

doi:10.1074/jbc.m401132200

Cited by 29 publications

(45 citation statements)

References 36 publications

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“…S1). The exact reasons are unknown for the apparent access of AMS to the lumi- nal side of IMVs, an observation with precedents (30,31). The simplest explanation may be that the small volume of the IMV lumen can be equilibrated rapidly even by slow diffusion of AMS across the membrane, although other possibilities such as translocation mediated by some channel-like membrane proteins and incomplete sealing of IMVs after the cell disruption cannot be excluded.…”

Section: Ams Modification Assay To Probe the Environments Of The Engimentioning

confidence: 99%

Environment of the Active Site Region of RseP, an Escherichia coli Regulated Intramembrane Proteolysis Protease, Assessed by Site-directed Cysteine Alkylation

Koide

Maegawa

Ito

et al. 2007

Journal of Biological Chemistry

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Regulated intramembrane proteolysis (RIP) plays crucial roles in both prokaryotic and eukaryotic organisms. Proteases for RIP cleave transmembrane regions of substrate membrane proteins. However, the molecular mechanisms for the proteolysis of membrane-embedded transmembrane sequences are largely unknown. Here we studied the environment surrounding the active site region of RseP, an Escherichia coli S2P ortholog involved in the E pathway of extracytoplasmic stress responses. RseP has two presumed active site motifs, HEXXH and LDG, located in membrane-cytoplasm boundary regions. We examined the reactivity of cysteine residues introduced within or in the vicinity of these two active site motifs with membrane-impermeable thiol-alkylating reagents under various conditions. The active site positions were inaccessible to the reagents in the native state, but many of them became partially modifiable in the presence of a chaotrope, while requiring simultaneous addition of a chaotrope and a detergent for full modification. These results suggest that the active site of RseP is not totally embedded in the lipid phase but located within a proteinaceous structure that is partially exposed to the aqueous milieu.Proteases contribute to functional regulation of some membrane proteins by introducing specific cleavages into them. For example, eukaryotic membrane-bound proteases such as ADAMs (a disintegrin and metalloproteinase) catalyze ectodomain shedding in which they cleave off extracellular domains of membrane proteins, including cytokines, growth factors, and adhesion molecules (1, 2). Membrane-embedded proteases are also involved in regulated intramembrane proteolysis (RIP) 2 , in which regulated cleavages are introduced into transmembrane segments of membrane proteins. An increasing number of findings have revealed that RIP plays pivotal roles in cell regulation and transmembrane signaling (3, 4). Proteases engaged in RIP include the S2P protease ␥-secretase, the rhomboid protease, and the signal peptide peptidase (3, 4). The S2P family proteases are widely distributed from bacteria to higher organisms and involved in diverse biological processes (5, 6). They possess conserved amino acid sequence motifs, a typical zinc metalloprotease motif HEXXH, and a C-terminal-located LDG motif (5, 6). Mutational alterations of the conserved residues in these motifs abolish the proteolytic activities (7-9), in agreement with the prediction that these motifs constitute a protease active site (10). The mammalian S2P protease, a founding member of RIP proteases, is an integral component of the regulatory systems for sterol metabolism and endoplasmic reticulum stress responses (11,12). In prokaryotes, two S2P homologs of Bacillus subtilis, SpoIVFB (13) and YluC (14), control sporulation and environmental stress responses, respectively. Caulobacter crescentus MmpA participates in cell polarity determination (15). Recent studies showed that S2P homologs of Vibrio cholerae and Mycobacterium tuberculosis (YaeL and Rv2869c, respectively) are i...

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Section: Ams Modification Assay To Probe the Environments Of The Engimentioning

confidence: 99%

Environment of the Active Site Region of RseP, an Escherichia coli Regulated Intramembrane Proteolysis Protease, Assessed by Site-directed Cysteine Alkylation

Koide

Maegawa

Ito

et al. 2007

Journal of Biological Chemistry

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“…This work identified Pro 145 , a part of the highly conserved TDP motif, to be implicated in the determination of transport properties of yeast plasma membrane antiporters, as far as substrate specificity range and transport activity are concerned. The fifth transmembrane domain of yeast antiporters can form a part of the substrate ion pathway (hydrophilic pore) similarly as was suggested for the fourth transmembrane segment in prokaryotic NhaA antiporters (62,68). Hydroxyl groups present in the side chains of amino acids might play a considerable role in the recognition of substrates as preliminary analysis of the other ZrSod2-22p versions obtained by random PCR mutagenesis suggest.…”

Section: Citatdpvlamentioning

confidence: 70%

“…1B). The binding of lithium cations, possessing the smallest atomic radius of all alkali metal cations, was suggested to cause more extensive conformational changes than the binding of a larger cation (62). On the other hand, the Li ϩ cation has the largest hydrated radius (0.340 nm), which can influence its ability to enter the binding site compared with other alkali metal cations (K ϩ , 0.232 nm; Rb ϩ , 0.228 nm).…”

Section: Citatdpvlamentioning

confidence: 99%

“…An acidic residue can be found at this position in all types of Na ϩ /H ϩ antiporters, from prokaryotes to mammals (30). Mutations of this aspartate significantly reduce the activity of the S. pombe sod2 antiporter (29,30) and of several bacterial antiporters (62)(63)(64)(65). In contrast to yeast Nha proteins, bacterial Na ϩ /H ϩ antiporters do not contain proline next to the aspartate, but there is another prolyl residue in the aspartate neighborhood (Pro 129 , the fourth residue preceding the important aspartate) that has been shown to be vital for E. coli NhaA function (65).…”

Section: Citatdpvlamentioning

confidence: 99%

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Identification of Conserved Prolyl Residue Important for Transport Activity and the Substrate Specificity Range of Yeast Plasma Membrane Na+/H+ Antiporters

Zimmermannová

Zavřel

Sychrová

2005

Journal of Biological Chemistry

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Yeast plasma membrane Na؉ /H ؉ antiporters are divided according to their substrate specificity in two distinct subfamilies. To identify amino acid residues responsible for substrate specificity determination (recognition of K ؉ ), the Zygosaccharomyces rouxii Sod2-22 antiporter (non-transporting K ؉ ) was mutagenized and a collection of ZrSod2-22 mutants that improved the KCl tolerance of a salt-sensitive Saccharomyces cerevisiae strain was isolated. Several independent ZrSod2-22 mutated alleles contained the replacement of a highly conserved proline 145 with a residue containing a hydroxyl group (Ser, Thr). Sitedirected mutagenesis of Pro 145 proved that an amino acid with a hydroxyl group at this position is enough to enable ZrSod2-22p to transport K ؉ . Simultaneously, the P145(S/T) mutation decreased the antiporter transport activity for both Na ؉ and Li ؉ . Replacement of Pro 145 with glycine resulted in a ZrSod2-22p with extremely low activity only for Na ؉ , and the exchange of a charged residue (Asp, Lys) for Pro 145 completely stopped the activity. Mutagenesis of the corresponding proline in the S. cerevisiae Nha1 antiporter (Pro 146 ) confirmed that this proline of the fifth transmembrane domain is a critical residue for antiporter function. This is the first evidence that a non-polar amino acid residue is important for the substrate specificity and activity of yeast Nha antiporters.

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“…The residues equivalent to these aspartates in NhaA, Asp163 and Asp164 (Supplementary Fig. 2 and 7) likely coordinate ion binding based on their position, conservation with mammalian Na + /H + antiporters 2 , phenotypes of mutants 1,21 , isothermal titration calorimetry (ITC) experiments 9 and MD simulations 8 . In NapA, mutation of either residue to alanine 15 or asparagine abolishes transport activity completely ( Supplementary Fig.…”

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confidence: 99%

A two-domain elevator mechanism for sodium/proton antiport

Lee¹,

Kang²,

Ballmoos³

et al. 2014

Acta Cryst Sect A

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Sodium/proton (Na + /H + ) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis 1 . 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 , where both EM and crystal structures are available [4][5][6] . NhaA is made up of two distinct domains, a Core domain and a Dimerisation 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 . Like many Na + /H + antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, where a conformational change is thought to occur 7 . To date, the only reported NhaA crystal structure 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 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the Core and Dimerisation 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 directly coordinate ion-binding 1,8,9 , 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, Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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The Fourth Transmembrane Domain of the Helicobacter pylori Na+/H+ Antiporter NhaA Faces a Water-filled Channel Required for Ion Transport

Cited by 29 publications

References 36 publications

Environment of the Active Site Region of RseP, an Escherichia coli Regulated Intramembrane Proteolysis Protease, Assessed by Site-directed Cysteine Alkylation

Environment of the Active Site Region of RseP, an Escherichia coli Regulated Intramembrane Proteolysis Protease, Assessed by Site-directed Cysteine Alkylation

Identification of Conserved Prolyl Residue Important for Transport Activity and the Substrate Specificity Range of Yeast Plasma Membrane Na+/H+ Antiporters

A two-domain elevator mechanism for sodium/proton antiport

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