Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na+/H+ antiporter NHA2

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F67985823%3A_____%2F22%3A00564736" target="_blank" >RIV/67985823:_____/22:00564736 - isvavai.cz</a>

Výsledek na webu

<a href="https://doi.org/10.1002/pro.4460" target="_blank" >https://doi.org/10.1002/pro.4460</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1002/pro.4460" target="_blank" >10.1002/pro.4460</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na+/H+ antiporter NHA2

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

Allosteric links between the hydrophilic N-terminus and transmembrane core of human Na+/H+ antiporter NHA2

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/GA21-08985S" target="_blank" >GA21-08985S: Eukaryotní antiportery Na+/H+ - klíčové prvky jejich struktury určující aktivitu, biogenezi a fyziologické funkce</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2022

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Protein Science

ISSN

0961-8368

e-ISSN

1469-896X

Svazek periodika

31

Číslo periodika v rámci svazku

12

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

22

Strana od-do

e4460

Kód UT WoS článku

000884401200001

EID výsledku v databázi Scopus

2-s2.0-85143088259