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Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na+/H+ antiporter NHA2
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SYSNO ASEP 0564736 Druh ASEP J - Článek v odborném periodiku Zařazení RIV J - Článek v odborném periodiku Poddruh J Článek ve WOS Název Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na+/H+ antiporter NHA2 Tvůrce(i) Velázquez, Diego (FGU-C)
Průša, Vojtěch (FGU-C)
Masrati, G. (IL)
Yariv, E. (IL)
Sychrová, Hana (FGU-C) RID, ORCID
Ben-Tal, N. (IL)
Zimmermannová, Olga (FGU-C) RID, ORCIDČíslo článku e4460 Zdroj.dok. Protein Science. - : Wiley - ISSN 0961-8368
Roč. 31, č. 12 (2022)Poč.str. 22 s. Jazyk dok. eng - angličtina Země vyd. US - Spojené státy americké Klíč. slova human NHA2 ; Na+/H+ antiporter ; N-terminal auto-inhibition ; phloretin ; yeast Obor OECD Biochemistry and molecular biology CEP GA21-08985S GA ČR - Grantová agentura ČR Způsob publikování Omezený přístup Institucionální podpora FGU-C - RVO:67985823 UT WOS 000884401200001 EID SCOPUS 85143088259 DOI 10.1002/pro.4460 Anotace The human Na+/H+ antiporter NHA2 (SLC9B2) transports Na+ or Li+ across the plasma membrane in exchange for protons, and is implicated in various pathologies. It is a 537 amino acids protein with an 82 residues long hydrophilic cytoplasmic N-terminus followed by a transmembrane part comprising 14 transmembrane helices. We optimized the functional expression of HsNHA2 in the plasma membrane of a salt-sensitive Saccharomyces cerevisiae strain and characterized in vivo a set of mutated or truncated versions of HsNHA2 in terms of their substrate specificity, transport activity, localization, and protein stability. We identified a highly conserved proline 246, located in the core of the protein, as being crucial for ion selectivity. The replacement of P246 with serine or threonine resulted in antiporters with altered substrate specificity that were not only highly active at acidic pH 4.0 (like the native antiporter), but also at neutral pH. P246T/S versions also exhibited increased resistance to the HsNHA2-specific inhibitor phloretin. We experimentally proved that a putative salt bridge between E215 and R432 is important for antiporter function, but also structural integrity. Truncations of the first 50-70 residues of the N-terminus doubled the transport activity of HsNHA2, while changes in the charge at positions E47, E56, K57, or K58 decreased the antiporter's transport activity. Thus, the hydrophilic N-terminal part of the protein appears to allosterically auto-inhibit cation transport of HsNHA2. Our data also show this in vivo approach to be useful for a rapid screening of SNP's effect on HsNHA2 activity. Pracoviště Fyziologický ústav Kontakt Lucie Trajhanová, lucie.trajhanova@fgu.cas.cz, Tel.: 241 062 400 Rok sběru 2023 Elektronická adresa https://doi.org/10.1002/pro.4460
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