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A twin histidine motif is the core structure for high-affinity substrate selection in plant ammonium transporters.

ABSTRACT: Ammonium transporters (AMT), methylamine permeases (Mep), and the more distantly related rhesus factors (Rh) are trimeric membrane proteins present in all domains of life. AMT/Mep/Rhs are highly selective membrane proteins required for ammonium uptake or release, and they efficiently exclude the similarly sized K⁺ ion. Previously reported crystal structures have revealed that each transporter subunit contains a unique hydrophobic but occluded central pore, but it is unclear whether the base (NH₃) or NH₃ coupled with an H⁺ are transported. Here, using expression of two plant AMTs (AtAMT1;2 and AMT2) in budding yeast, we found that systematic replacements in the conserved twin-histidine motif, a hallmark of most AMT/Mep/Rh, alter substrate recognition, transport capacities, N isotope selection, and selectivity against K⁺ AMT-specific differences were found for histidine variants. Variants that completely lost ammonium N isotope selection, a feature likely associated with NH₄ ⁺ deprotonation during passage, substantially transported K⁺ in addition to NH₄ ⁺ Of note, the twin-histidine motif was not essential for ammonium transport. However, it conferred key AMT features, such as high substrate affinity and selectivity against alkali cations via an NH₄ ⁺ deprotonation mechanism. Our findings indicate that the twin-His motif is the core structure responsible for substrate deprotonation and isotopic preferences in AMT pores and that decreased deprotonation capacity is associated with reduced selectivity against K⁺ We conclude that optimization for ammonium transport in plant AMT represents a compromise between substrate deprotonation for optimal selectivity and high substrate affinity and transport rates.

SUBMITTER: Ganz P

PROVIDER: S-EPMC7062152 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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A twin histidine motif is the core structure for high-affinity substrate selection in plant ammonium transporters.

Ganz Pascal P Ijato Toyosi T Porras-Murrilo Romano R Stührwohldt Nils N Ludewig Uwe U Neuhäuser Benjamin B

The Journal of biological chemistry 20200127 10

Ammonium transporters (AMT), methylamine permeases (Mep), and the more distantly related rhesus factors (Rh) are trimeric membrane proteins present in all domains of life. AMT/Mep/Rhs are highly selective membrane proteins required for ammonium uptake or release, and they efficiently exclude the similarly sized K<sup>+</sup> ion. Previously reported crystal structures have revealed that each transporter subunit contains a unique hydrophobic but occluded central pore, but it is unclear whether th ...[more]

PMID: 31988244

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Project description:The importance of the family of high affinity ammonium transporters is demonstrated by the presence of these proteins in all domains of life, including bacteria, archaea, fungi, plants, and humans. The majority of the proteins that have been studied from this family show high affinity and selectivity for ammonium, are impermeable to alkaline cations, saturate rapidly at low millimolar concentrations and most of them, are also permeable to methylammonium. Crystallization of homologue proteins from bacteria and archaea has demonstrated that the functional entity corresponds to a trimer, with each monomer maintaining a conductive pore. Through molecular modeling, it has been demonstrated that even though the identity of the proteins between bacteria/archaea with those from plants is below 25%, the latter seem to maintain similar tertiary and quaternary structures, an observation that has helped to address the functionality of conserved residues by means of mutational analysis. Results have shown that changes in the extracellular binding site of some plant transporters may result in their inhibition or reduction in transport activity, while in Escherichia coli, dissimilar replacements like Phe/Ala or Ser/Leu that eliminate possible π-interactions or H-bonds with ammonium, respectively, lead to more active transporters. Active mutants with changes in the pair of conserved His in the center of the transporter suggest these residues are dispensable. Additional mutations have identified other important amino acids, both in the entrance of the pore and in cytoplasmic loops. Regulation of this family of transporters can be achieved by interactions of the C-terminal with cytoplasmic loops within the same monomer, or with a neighbor in the trimer. Depending on the interacting residues, these contacts may lead to the activation or inhibition of the protein. The aim of this review is to critically evaluate the newest findings on the role of the proposed amino acids that structure the ammonium pathway, as well as highlight the importance of additional residues that have been identified through mutational analyses.

Dataset Information

A twin histidine motif is the core structure for high-affinity substrate selection in plant ammonium transporters.

Publications

A twin histidine motif is the core structure for high-affinity substrate selection in plant ammonium transporters.

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