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Structural and functional insights into the mechanism of action of plant borate transporters.

ABSTRACT: Boron has essential roles in plant growth and development. BOR proteins are key in the active uptake and distribution of boron, and regulation of intracellular boron concentrations. However, their mechanism of action remains poorly studied. BOR proteins are homologues of the human SLC4 family of transporters, which includes well studied mammalian transporters such as the human Anion Exchanger 1 (hAE1). Here we generated Arabidopsis thaliana BOR1 (AtBOR1) variants based (i) on known disease causing mutations of hAE1 (S466R, A500R) and (ii) a loss of function mutation (D311A) identified in the yeast BOR protein, ScBOR1p. The AtBOR1 variants express in yeast and localise to the plasma membrane, although both S466R and A500R exhibit lower expression than the WT AtBOR1 and D311A. The D311A, S466R and A500R mutations result in a loss of borate efflux activity in a yeast bor1p knockout strain. A. thaliana plants containing these three individual mutations exhibit substantially decreased growth phenotypes in soil under conditions of low boron. These data confirm an important role for D311 in the function of the protein and show that mutations equivalent to disease-causing mutations in hAE1 have major effects in AtBOR1. We also obtained a low resolution cryo-EM structure of a BOR protein from Oryza sativa, OsBOR3, lacking the 30 C-terminal amino acid residues. This structure confirms the gate and core domain organisation previously observed for related proteins, and is strongly suggestive of an inward facing conformation.

SUBMITTER: Saouros S

PROVIDER: S-EPMC8192573 | biostudies-literature | 2021 Jun

REPOSITORIES: biostudies-literature

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Structural and functional insights into the mechanism of action of plant borate transporters.

Saouros Savvas S Mohan Thotegowdanapalya C TC Cecchetti Cristina C Lehmann Silke S Barrit Joseph D JD Scull Nicola J NJ Simpson Paul P Alguel Yilmaz Y Cameron Alexander D AD Jones Alexandra M E AME Byrne Bernadette B

Scientific reports 20210610 1

Boron has essential roles in plant growth and development. BOR proteins are key in the active uptake and distribution of boron, and regulation of intracellular boron concentrations. However, their mechanism of action remains poorly studied. BOR proteins are homologues of the human SLC4 family of transporters, which includes well studied mammalian transporters such as the human Anion Exchanger 1 (hAE1). Here we generated Arabidopsis thaliana BOR1 (AtBOR1) variants based (i) on known disease causi ...[more]

PMID: 34112901

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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.

Project description:BackgroundThe Arabidopsis thaliana MHX gene (AtMHX) encodes a Mg²⁺/H⁺ exchanger. Among non-plant proteins, AtMHX showed the highest similarity to mammalian Na⁺/Ca²⁺ exchanger (NCX) transporters, which are part of the Ca²⁺/cation (CaCA) exchanger superfamily.ResultsSequences showing similarity to AtMHX were searched in the databases or sequenced from cDNA clones. Phylogenetic analysis showed that the MHX family is limited to plants, and constitutes a sixth family within the CaCA superfamily. Some plants include, besides a full MHX gene, partial MHX-related sequences. More than one full MHX gene was currently identified only in Oryza sativa and Mimulus guttatus, but an EST for more than one MHX was identified only in M. guttatus. MHX genes are not present in the currently available chlorophyte genomes. The prevalence of upstream ORFs in MHX genes is much higher than in most plant genes, and can limit their expression. A structural model of the MHXs, based on the resolved structure of NCX1, implies that the MHXs include nine transmembrane segments. The MHXs and NCXs share 32 conserved residues, including a GXG motif implicated in the formation of a tight-turn in a reentrant-loop. Three residues differ between all MHX and NCX proteins. Altered mobility under reducing and non-reducing conditions suggests the presence of an intramolecular disulfide-bond in AtMHX.ConclusionsThe absence of MHX genes in non-plant genomes and in the currently available chlorophyte genomes, and the presence of an NCX in Chlamydomonas, are consistent with the suggestion that the MHXs evolved from the NCXs after the split of the chlorophyte and streptophyte lineages of the plant kingdom. The MHXs underwent functional diploidization in most plant species. De novo duplication of MHX occurred in O. sativa before the split between the Indica and Japonica subspecies, and was apparently followed by translocation of one MHX paralog from chromosome 2 to chromosome 11 in Japonica. The structural analysis presented and the identification of elements that differ between the MHXs and the NCXs, or between the MHXs of specific plant groups, can contribute to clarification of the structural basis of the function and ion selectivity of MHX transporters.

Dataset Information

Structural and functional insights into the mechanism of action of plant borate transporters.

Publications

Structural and functional insights into the mechanism of action of plant borate transporters.

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