Function of Transmembrane Domain IX in the Na+/Proline Transporter PutP

Raba, Michael; Baumgärtner, Tobias; Hilger, Daniel; Klempahn, Katrin; Härtel, Tobias; Jung, Kirsten; Jung, Heinrich

doi:10.1016/j.jmb.2008.07.070

Cited by 18 publications

(44 citation statements)

References 40 publications

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“…2). Binding to this site is affected by mutagenesis of S365 in vSGLT to alanine and of analogous residues in other SSS proteins (29)(30)(31). Consistent with the observations for Mhp1, MD simulations of these inward-facing conformations of vSGLT show that ions are released quickly from the proposed binding site (28,(32)(33)(34)(35).…”

supporting

confidence: 67%

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Khafizov¹,

Pérez²,

Koshy³

et al. 2012

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

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Sodium-coupled substrate transport plays a central role in many biological processes. However, despite knowledge of the structures of several sodium-coupled transporters, the location of the sodiumbinding site(s) often remains unclear. Several of these structures have the five transmembrane-helix inverted-topology repeat, LeuTlike (FIRL) fold, whose pseudosymmetry has been proposed to facilitate the alternating-access mechanism required for transport. Here, we provide biophysical, biochemical, and computational evidence for the location of the two cation-binding sites in the sodium-coupled betaine symporter BetP. A recent X-ray structure of BetP in a sodium-bound closed state revealed that one of these sites, equivalent to the Na2 site in related transporters, is located between transmembrane helices 1 and 8 of the FIRL-fold; here, we confirm the location of this site by other means. Based on the pseudosymmetry of this fold, we hypothesized that the second site is located between the equivalent helices 6 and 3. Molecular dynamics simulations of the closed-state structure suggest this second sodium site involves two threonine sidechains and a backbone carbonyl from helix 3, a phenylalanine from helix 6, and a water molecule. Mutating the residues proposed to form the two binding sites increased the apparent K m and K d for sodium, as measured by betaine uptake, tryptophan fluorescence, and 22 Na + binding, and also diminished the transient currents measured in proteoliposomes using solid supported membrane-based electrophysiology. Taken together, these results provide strong evidence for the identity of the residues forming the sodium-binding sites in BetP.secondary transport | symmetry | membrane protein | alkali metal ion | osmoregulation

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supporting

confidence: 67%

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Khafizov¹,

Pérez²,

Koshy³

et al. 2012

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

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“…A recent study of PutP using the substituted cysteine-accessibility method (SCAM) supports the presence of the S c site (42). In this study, labeling of the individual thiol group at residue positions 344, 347, 348, and 351 was reduced in the presence of the substrate proline but not by Na ϩ alone (42).…”

Section: Crystallographically Identified Substrate-binding Sites In Vmentioning

confidence: 50%

“…The C␣-C␤ bonds of substrate binding residues are shown as sticks (cyan for the S1/S c site and green for the S2/S e sites) with the C␤ atoms represented as small spheres. D, residues Ser-365, Ser-368, and Ser-372 of vSGLT are aligned to Thr-341, Cys-344, and Val-348 of PutP, which have been found to be critical for proline uptake (42).…”

Section: Structure-structure Alignment Between Leut and Vsglt-mentioning

confidence: 99%

Identification of a Second Substrate-binding Site in Solute-Sodium Symporters

Zheng

Lee

Bracher

et al. 2015

Journal of Biological Chemistry

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Background: Although the solute-sodium symporter (SSS) vSGLT and the neurotransmitter-sodium symporter (NSS) LeuT have similar structural folds, their crystallographically identified substrate sites diverge in location and composition. Results: We identified second substrate sites in two SSSs that align with the crystallographically identified site in LeuT. Conclusion: Substrate transport by SSSs involves two substrate sites. Significance: NSS and SSS share common mechanistic features.

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“…1). The transmembrane domain is the important topology determined domain of membrane protein, 21 all conserved amino acids residues were selected for site-directed mutagenesis according to the length of side chain, with charge or not, and amino acid hydrophobicity. The whole alterations (C150W, E155A, G158E, L182F, P186R, F220L, D224A, Q231H, A232D, G234S, G235R, G258C, Q262H, D382G, F396L, and F402V) were introduced into the vector pBES116 and the mutant plasmids were transformed to competent cells.…”

Section: Construction and Expression Of Mutant Rta2 Reintegration In mentioning

confidence: 99%

Mutation of G234 amino acid residue inCandida albicansdrug-resistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport

Zhang

Miao

et al. 2015

Virulence

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(2015) Mutation of G234 amino acid residue in Candidaalbicans drugresistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport, Virulence, 6:6, 599-607, DOI: 10.1080/21505594.2015 Widespread and repeated use of azoles has led to the rapid development of drug resistance in Candida albicans. Our previous study found Rta2p, a membrane protein with 7 transmembrane domains, was involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Conserved amino acids in the transmembrane domain of Rta2p were subjected to site-directed mutagenesis. The sensitivity of C. albicans to fluconazole in vitro was examined by minimum inhibitory concentration and killing assay, and the therapeutic efficacy of fluconazole in vivo was performed by systemic mice candidiasis model. Furthermore, dihydrosphingosine transport activity was detected by NBD labeled D-erythro-dihydrosphingosine uptake and release assay, and the sensitivity to sphingolipid biosynthesis inhibitors. We successfully constructed 14 mutant strains of Rta2p, screened them by minimum inhibitory concentration and found Ca 2C did not completely induce fluconazole resistance with G158E and G234S mutations. Furthermore, we confirmed that G234S mutant enhanced the therapeutic efficacy of fluconazole against systemic candidiasis and significantly increased the accumulation of dihydrosphingosine by decreasing its release. However, G158E mutant didn't affect drug therapeutic efficacy in vivo and dihydrosphingosine transport in C. albicans. G234 of Rta2p in C. albicans is crucial in calcineurin-mediated fluconazole resistance and dihydrosphingosine transport.

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Function of Transmembrane Domain IX in the Na+/Proline Transporter PutP

Cited by 18 publications

References 40 publications

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Investigation of the sodium-binding sites in the sodium-coupled betaine transporter BetP

Identification of a Second Substrate-binding Site in Solute-Sodium Symporters

Mutation of G234 amino acid residue inCandida albicansdrug-resistance-related protein Rta2p is associated with fluconazole resistance and dihydrosphingosine transport

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