Site-directed mutagenesis at the Glu78 in Ec-NhaA transporter impacting ion exchange: a biophysical study.

Na+/H+ antiporters facilitate the exchange of Na+ for H+ across the cytoplasmic membrane in prokaryotic and eukaryotic cells. These transporters are crucial to maintain the homeostasis of sodium ions, consequently pH, and volume of the cells. Therefore, sodium/proton antiporters are considered promising therapeutic targets in humans. The Na+/H+ antiporter in Escherichia coli (Ec-NhaA), a prototype of cation-proton antiporter (CPA) family, transports two protons and one sodium (or Li+) in opposite direction. Previous mutagenesis experiments on Ec-NhaA have proposed Asp164, Asp163, and Asp133 amino acids with the significant implication in functional and structural integrity and create site for ion-binding. However, the mechanism and the sites for the binding of the two protons remain unknown and controversial which could be critical for pH regulation. In this study, we have explored the role of Glu78 in the regulation of pH by Ec-NhaA. Although we have created various mutants, E78C has shown a considerable effect on the stoichiometry of NhaA and presented comparable phenotypes. The ITC experiment has shown the binding of ~ 5 protons in response to the transport of one lithium ion. The phenotype analysis on selective medium showed a significant expression compared to WT Ec-NhaA. This represents the importance of Glu78 in transporting the H+ across the membrane where a single mutation with Cys amino acid alters the number of H+ significantly maintaining the activity of the protein.

The molecular sociology of NHERF1 PDZ proteins controlling renal hormone-regulated phosphate transport.

Parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) control extracellular phosphate levels by regulating renal NPT2A-mediated phosphate transport by a process requiring the PDZ scaffold protein NHERF1. NHERF1 possesses two PDZ domains, PDZ1 and PDZ2, with identical core-binding GYGF motifs explicitly recognizing distinct binding partners that play different and specific roles in hormone-regulated phosphate transport. The interaction of PDZ1 and the carboxy-terminal PDZ-binding motif of NPT2A (C-TRL) is required for basal phosphate transport. PDZ2 is a regulatory domain that scaffolds multiple biological targets, including kinases and phosphatases involved in FGF23 and PTH signaling. FGF23 and PTH trigger disassembly of the NHERF1-NPT2A complex through reversible hormone-stimulated phosphorylation with ensuing NPT2A sequestration, down-regulation, and cessation of phosphate absorption. In the absence of NHERF1-NPT2A interaction, inhibition of FGF23 or PTH signaling results in disordered phosphate homeostasis and phosphate wasting. Additional studies are crucial to elucidate how NHERF1 spatiotemporally coordinates cellular partners to regulate extracellular phosphate levels.

The Hydrophilic C-terminus of Yeast Plasma-membrane Na+/H+ Antiporters Impacts Their Ability to Transport K.

Yeast plasma-membrane Na+/H+ antiporters (Nha/Sod) ensure the optimal intracellular level of alkali-metal cations and protons in cells. They are predicted to consist of 13 transmembrane segments (TMSs) and a large hydrophilic C-terminal cytoplasmic part with seven conserved domains. The substrate specificity, specifically the ability to recognize and transport K+ cations in addition to Na+ and Li+, differs among homologs. In this work, we reveal that the composition of the C-terminus impacts the ability of antiporters to transport particular cations. In the osmotolerant yeast Zygosaccharomyces rouxii, the Sod2-22 antiporter only efficiently exports Na+ and Li+, but not K+. The introduction of a negative charge or removal of a positive charge in one of the C-terminal conserved regions (C3) enabled ZrSod2-22 to transport K+. The same mutations rescued the low level of activity and purely Li+ specificity of ZrSod2-22 with the A179T mutation in TMS6, suggesting a possible interaction between this TMS and the C-terminus. The truncation or replacement of the C-terminal part of ZrSod2-22 with the C-terminus of a K+-transporting Nha/Sod antiporter (Saccharomyces cerevisiae Nha1 or Z. rouxii Nha1) also resulted in an antiporter with the capacity to export K+. In addition, in ScNha1, the replacement of three positively charged arginine residues 539-541 in the C3 region with alanine caused its inability to provide cells with tolerance to Li+. All our results demonstrate that the physiological functions of yeast Nha/Sod antiporters, either in salt tolerance or in K+ homeostasis, depend on the composition of their C-terminal parts.

Structural basis for the activity regulation of Salt Overly Sensitive 1 in Arabidopsis salt tolerance.

The plasma membrane Na+/H+ exchanger Salt Overly Sensitive 1 (SOS1) is crucial for plant salt tolerance. Unlike typical sodium/proton exchangers, SOS1 contains a large cytoplasmic domain (CPD) that regulates Na+/H+ exchange activity. However, the underlying modulation mechanism remains unclear. Here we report the structures of SOS1 from Arabidopsis thaliana in two conformations, primarily differing in CPD flexibility. The CPD comprises an interfacial domain, a cyclic nucleotide-binding domain-like domain (CNBD-like domain) and an autoinhibition domain. Through yeast cell-based Na+ tolerance test, we reveal the regulatory role of the interfacial domain and the activation role of the CNBD-like domain. The CPD forms a negatively charged cavity that is connected to the ion binding site. The transport of Na+ may be coupled with the conformational change of CPD. These findings provide structural and functional insight into SOS1 activity regulation.

Structure and electromechanical coupling of a voltage-gated Na+/H+ exchanger.

Voltage-sensing domains control the activation of voltage-gated ion channels, with a few exceptions1. One such exception is the sperm-specific Na+/H+ exchanger SLC9C1, which is the only known transporter to be regulated by voltage-sensing domains2-5. After hyperpolarization of sperm flagella, SLC9C1 becomes active, causing pH alkalinization and CatSper Ca2+ channel activation, which drives chemotaxis2,6. SLC9C1 activation is further regulated by cAMP2,7, which is produced by soluble adenyl cyclase (sAC). SLC9C1 is therefore an essential component of the pH-sAC-cAMP signalling pathway in metazoa8,9, required for sperm motility and fertilization4. Despite its importance, the molecular basis of SLC9C1 voltage activation is unclear. Here we report cryo-electron microscopy (cryo-EM) structures of sea urchin SLC9C1 in detergent and nanodiscs. We show that the voltage-sensing domains are positioned in an unusual configuration, sandwiching each side of the SLC9C1 homodimer. The S4 segment is very long, 90 Å in length, and connects the voltage-sensing domains to the cytoplasmic cyclic-nucleotide-binding domains. The S4 segment is in the up configuration-the inactive state of SLC9C1. Consistently, although a negatively charged cavity is accessible for Na+ to bind to the ion-transporting domains of SLC9C1, an intracellular helix connected to S4 restricts their movement. On the basis of the differences in the cryo-EM structure of SLC9C1 in the presence of cAMP, we propose that, upon hyperpolarization, the S4 segment moves down, removing this constriction and enabling Na+/H+ exchange.

Structures of a sperm-specific solute carrier gated by voltage and cAMP.

The newly characterized sperm-specific Na+/H+ exchanger stands out by its unique tripartite domain composition1,2. It unites a classical solute carrier unit with regulatory domains usually found in ion channels, namely, a voltage-sensing domain and a cyclic-nucleotide binding domain1,3, which makes it a mechanistic chimera and a secondary-active transporter activated strictly by membrane voltage. Our structures of the sea urchin SpSLC9C1 in the absence and presence of ligands reveal the overall domain arrangement and new structural coupling elements. They allow us to propose a gating model, where movements in the voltage sensor indirectly cause the release of the exchanging unit from a locked state through long-distance allosteric effects transmitted by the newly characterized coupling helices. We further propose that modulation by its ligand cyclic AMP occurs by means of disruption of the cytosolic dimer interface, which lowers the energy barrier for S4 movements in the voltage-sensing domain. As SLC9C1 members have been shown to be essential for male fertility, including in mammals2,4,5, our structure represents a potential new platform for the development of new on-demand contraceptives.

Mutations in an unrecognized internal NPT2A PDZ motif disrupt phosphate transport and cause congenital hypophosphatemia.

The Na+-dependent phosphate cotransporter-2A (NPT2A, SLC34A1) is a primary regulator of extracellular phosphate homeostasis. Its most prominent structural element is a carboxy-terminal PDZ ligand that binds Na+/H+ Exchanger Regulatory Factor-1 (NHERF1, SLC9A3R1). NHERF1, a multidomain PDZ protein, establishes NPT2A membrane localization and is required for hormone-inhibitable phosphate transport. NPT2A also possesses an uncharacterized internal PDZ ligand. Two recent clinical reports describe congenital hypophosphatemia in children harboring Arg495His or Arg495Cys variants within the internal PDZ motif. The wild-type internal 494TRL496 PDZ ligand binds NHERF1 PDZ2, which we consider a regulatory domain. Ablating the internal PDZ ligand with a 494AAA496 substitution blocked hormone-inhibitable phosphate transport. Complementary approaches, including CRISPR/Cas9 technology, site-directed mutagenesis, confocal microscopy, and modeling, showed that NPT2A Arg495His or Arg495Cys variants do not support PTH or FGF23 action on phosphate transport. Coimmunoprecipitation experiments indicate that both variants bind NHERF1 similarly to WT NPT2A. However, in contrast with WT NPT2A, NPT2A Arg495His, or Arg495Cys variants remain at the apical membrane and are not internalized in response to PTH. We predict that Cys or His substitution of the charged Arg495 changes the electrostatics, preventing phosphorylation of the upstream Thr494, interfering with phosphate uptake in response to hormone action, and inhibiting NPT2A trafficking. We advance a model wherein the carboxy-terminal PDZ ligand defines apical localization NPT2A, while the internal PDZ ligand is essential for hormone-triggered phosphate transport.

Phytomolecules as potential candidates to intervene the function of E. coli sodium-proton antiporters; Ec-NhaA.

Sodium-Proton antiporter, NhaA is a ubiquitous protein found in cytoplasmic membranes of all the prokaryotic and eukaryotic systems. These antiporters have been widely studied in E. coli and their homologs, observed in humans, are found to be crucial for various pathophysiological conditions, such as hypertension, cardiac diseases, blood pressure fluctuation etc. NhaA is responsible for the virulent properties of many pathogens like Vibrio cholerae, Yersinia pestis etc. In the present work, we have exploited in silico approaches to find lead phytomolecules that have the efficacy to interfere with the activities of sodium-proton antiporters in E. coli. A database of the plant-based natural bioactive compounds was used to screen 350 phytochemicals from various plant sources as potential ligands for the Ec-NhaA protein (PDB ID: 4ATV). Further interactions between Ec-NhaA and ligands were analyzed by AutoDock Vina and proposed 46 ligands with a significant affinity for NhaA where the binding energy range from -7.5 to -9.3 kcal/mol. Physiochemical characterization suggested 26 ligands with non-BBB permeability, good GI absorption and solubility. As a final step, MD simulation for more than 100 ns duration suggested Luteolin, Apigenin and Rhamnocitrin with the best affinity and showing potential stable interaction with the target protein. This study proposed the potential compounds of natural origin as an interfering agent against sodium-proton transport activity that may lead to affect the survival of various pathogenic bacteria.Communicated by Ramaswamy H. Sarma.

Altered distribution and localization of organellar Na+/H+ exchangers in postmortem schizophrenia dorsolateral prefrontal cortex.

Schizophrenia is a complex and multifactorial disorder associated with altered neurotransmission as well as numerous signaling pathway and protein trafficking disruptions. The pH of intracellular organelles involved in protein trafficking is tightly regulated and impacts their functioning. The SLC9A family of Na+/H+ exchangers (NHEs) plays a fundamental role in cellular and intracellular pH homeostasis. Four organellar NHE isoforms (NHE6-NHE9) are targeted to intracellular organelles involved in protein trafficking. Increased interactions between organellar NHEs and receptor of activated protein C kinase 1 (RACK1) can lead to redistribution of NHEs to the plasma membrane and hyperacidification of target organelles. Given their role in organelle pH regulation, altered expression and/or localization of organellar NHEs could be an underlying cellular mechanism contributing to abnormal intracellular trafficking and disrupted neurotransmitter systems in schizophrenia. We thus characterized organellar NHE expression, co-immunoprecipitation with RACK1, and Triton X-114 (TX-114) phase partitioning in dorsolateral prefrontal cortex of 25 schizophrenia and 25 comparison subjects by Western blot analysis. In schizophrenia after controlling for subject age at time of death, postmortem interval, tissue pH, and sex, there was significantly decreased total expression of NHE8, decreased co-immunoprecipitation of NHE8 (64%) and NHE9 (56%) with RACK1, and increased TX-114 detergent phase partitioning of NHE6 (283%), NHE9 (75%), and RACK1 (367%). Importantly, none of these dependent measures was significantly impacted when comparing those in the schizophrenia group on antipsychotics to those off of antipsychotics for at least 6 weeks at their time of death and none of these same proteins were affected in rats chronically treated with haloperidol. In summary, we characterized organellar NHE expression and distribution in schizophrenia DLPFC and identified abnormalities that could represent a novel mechanism contributing to disruptions in protein trafficking and neurotransmission in schizophrenia.

Ubiquitous Existence of Cation-Proton Antiporter and its Structurefunction Interplay: A Clinical Prospect.

Sodium, potassium, and protons are the most important ions for life on earth, and their homeostasis is crucially needed for the survival of cells. The biological cells have developed a system that regulates and maintains the integrity of the cells by facilitating the exchange of these ions. These systems include the specific type of ion transporter membrane proteins such as cation-proton antiporters. Cation proton antiporters induce the active transport of cations like Na+, K+ or Ca+ across the cell membrane in exchange for protons (H+) and make the organism able to survive in alkaline conditions, high or fluctuating pH, stressed temperature or osmolarity. The secondary transporter proteins exploit the properties of various specific structural components to carry out efficient active transport. Ec-NhaA crystal structure was resolved at acidic pH at which the protein is downregulated, which discloses the presence of 12 transmembrane (TM) helices. This structural fold, the "NhaA fold," is speculated to contribute to the cation-binding site and conformational alterations during transport in various antiporters. Irrespective of the variation in the composition of amino acids and lengths of proteins, several other members of the CPA family, such as NmABST, PaNhaP, and MjNhaP1, share the common structural features of the Ec-NhaA. The present review elucidates the existence of CPAs throughout all the kingdoms and the structural intercorrelation with their function. The interplay in the structurefunction of membrane transporter protein may be implemented to explore the plethora of biological events such as conformation, folding, ion binding and translocation etc.

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

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.

The pH sensor and ion binding of NhaD Na+ /H+ antiporter from IT superfamily.

Sodium-proton (Na+ /H+ ) antiporters from the ion transporter (IT) superfamily play a vital role in controlling the pH and electrolyte homeostasis. However, very limited information regarding their structural functions is available to date. In this study, the structural model of the NhaD antiporter was proposed as a typical hairpin structure of IT proteins, with two symmetrically conserved scaffold domains that frame the core substrate-binding sites, and four motifs were identified. Furthermore, 25 conserved sites involving these domains were subjected to site-directed mutagenesis, and all mutations resulted in an impact on transport abilities. In particular, as candidates for Na+ -binding sites, D166 and D405 mutations at hairpin discontinuities were detrimental to transport activities and were found to induce pronounced conformational changes using fluorescence resonance energy transfer (FRET) assays. In addition, as observed in the NhaA structure, some charged residues, for example, E64, E65, R454, and R464, are predicted to be involved in the net charge switch of NhaD activation, by collectively form a "pH sensor" at the entrance of the cytoplasmic funnel. Mutations encompassing these residues were detrimental to the transport activity of NhaD or lost the capacity to respond to pH signals and trigger conformational changes for Na+ translocation.

Structure, mechanism and lipid-mediated remodeling of the mammalian Na+/H+ exchanger NHA2.

The Na+/H+ exchanger SLC9B2, also known as NHA2, correlates with the long-sought-after Na+/Li+ exchanger linked to the pathogenesis of diabetes mellitus and essential hypertension in humans. Despite the functional importance of NHA2, structural information and the molecular basis for its ion-exchange mechanism have been lacking. Here we report the cryo-EM structures of bison NHA2 in detergent and in nanodiscs, at 3.0 and 3.5 Å resolution, respectively. The bison NHA2 structure, together with solid-state membrane-based electrophysiology, establishes the molecular basis for electroneutral ion exchange. NHA2 consists of 14 transmembrane (TM) segments, rather than the 13 TMs previously observed in mammalian Na+/H+ exchangers (NHEs) and related bacterial antiporters. The additional N-terminal helix in NHA2 forms a unique homodimer interface with a large intracellular gap between the protomers, which closes in the presence of phosphoinositol lipids. We propose that the additional N-terminal helix has evolved as a lipid-mediated remodeling switch for the regulation of NHA2 activity.

Rare variant analysis in eczema identifies exonic variants in DUSP1, NOTCH4 and SLC9A4.

Previous genome-wide association studies revealed multiple common variants involved in eczema but the role of rare variants remains to be elucidated. Here, we investigate the role of rare variants in eczema susceptibility. We meta-analyze 21 study populations including 20,016 eczema cases and 380,433 controls. Rare variants are imputed with high accuracy using large population-based reference panels. We identify rare exonic variants in DUSP1, NOTCH4, and SLC9A4 to be associated with eczema. In DUSP1 and NOTCH4 missense variants are predicted to impact conserved functional domains. In addition, five novel common variants at SATB1-AS1/KCNH8, TRIB1/LINC00861, ZBTB1, TBX21/OSBPL7, and CSF2RB are discovered. While genes prioritized based on rare variants are significantly up-regulated in the skin, common variants point to immune cell function. Over 20% of the single nucleotide variant-based heritability is attributable to rare and low-frequency variants. The identified rare/low-frequency variants located in functional protein domains point to promising targets for novel therapeutic approaches to eczema.

PDZ-Containing Proteins Targeted by the ACE2 Receptor.

Angiotensin-converting enzyme 2 (ACE2) is a main receptor for SARS-CoV-2 entry to the host cell. Indeed, the first step in viral entry is the binding of the viral trimeric spike (S) protein to ACE2. Abundantly present in human epithelial cells of many organs, ACE2 is also expressed in the human brain. ACE2 is a type I membrane protein with an extracellular N-terminal peptidase domain and a C-terminal collectrin-like domain that ends with a single transmembrane helix and an intracellular 44-residue segment. This C-terminal segment contains a PDZ-binding motif (PBM) targeting protein-interacting domains called PSD-95/Dlg/ZO-1 (PDZ). Here, we identified the human PDZ specificity profile of the ACE2 PBM using the high-throughput holdup assay and measuring the binding intensities of the PBM of ACE2 against the full human PDZome. We discovered 14 human PDZ binders of ACE2 showing significant binding with dissociation constants' values ranging from 3 to 81 µM. NHERF, SHANK, and SNX27 proteins found in this study are involved in protein trafficking. The PDZ/PBM interactions with ACE2 could play a role in ACE2 internalization and recycling that could be of benefit for the virus entry. Interestingly, most of the ACE2 partners we identified are expressed in neuronal cells, such as SHANK and MAST families, and modifications of the interactions between ACE2 and these neuronal proteins may be involved in the neurological symptoms of COVID-19.

Functional Dynamics of an Ancient Membrane-Bound Hydrogenase.

The membrane-bound hydrogenase (Mbh) is a redox-driven Na+/H+ transporter that employs the energy from hydrogen gas (H2) production to catalyze proton pumping and Na+/H+ exchange across cytoplasmic membranes of archaea. Despite a recently resolved structure of this ancient energy-transducing enzyme [Yu et al. Cell 2018, 173, 1636-1649], the molecular principles of its redox-driven ion-transport mechanism remain puzzling and of major interest for understanding bioenergetic principles of early cells. Here we use atomistic molecular dynamics (MD) simulations in combination with data clustering methods and quantum chemical calculations to probe principles underlying proton reduction as well as proton and sodium transport in Mbh from the hyperthermophilic archaeon Pyrococcus furiosus. We identify putative Na+ binding sites and proton pathways leading across the membrane and to the NiFe-active center as well as conformational changes that regulate ion uptake. We suggest that Na+ binding and protonation changes at a putative ion-binding site couple to proton transfer across the antiporter-like MbhH subunit by modulating the conformational state of a conserved ion pair at the subunit interface. Our findings illustrate conserved coupling principles within the complex I superfamily and provide functional insight into archaeal energy transduction mechanisms.

Towards Molecular Understanding of the pH Dependence Characterizing NhaA of Which Structural Fold is Shared by Other Transporters.

Na+/H+ antiporters comprise a super-family (CPA) of membrane proteins that are found in all kingdoms of life and are essential in cellular homeostasis of pH, Na+ and volume. Their activity is strictly dependent on pH, a property that underpins their role in pH homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystal structure provided insight into the architecture of this molecular machine. However, the mechanism of the strict pH dependence of NhaA is missing. Here, as a follow up of a recent evolutionary analysis that identified a 'CPA motif', we rationally designed three E. coli NhaA mutants: D133S, I134T, and the double mutant D133S-I134T. Exploring growth phenotype, transport activity and Li+-binding of the mutants, we revealed that Asp133 does not participate directly in proton binding, nor does it directly dictate the pH-dependent transport of NhaA. Strikingly, the variant I134T lost some of the pH control, and the D133S-Il134T double mutant retained Li+ binding in a pH independent fashion. Concurrent to loss of pH control, these mutants bound Li+ more strongly than the WT. Both positions are in close vicinity to the ion-binding site of the antiporter, attributing the results to electrostatic interaction between these residues and Asp164 of the ion-binding site. This is consistent with pH sensing resulting from direct coupling between cation binding and deprotonation in Asp164, which applies also to other CPA antiporters that are involved in human diseases.

GhNHX3D, a Vacuolar-Localized Na+/H+ Antiporter, Positively Regulates Salt Response in Upland Cotton.

Vacuolar sodium/proton (Na+/H+) antiporters (NHXs) can stabilize ion contents to improve the salt tolerance of plants. Here, GhNHX3D was cloned and characterized from upland cotton (Gossypium hirsutum). Phylogenetic and sequence analyses showed that GhNHX3D belongs to the vacuolar-type NHXs. The GhNHX3D-enhanced green fluorescent protein (eGFP) fusion protein localized on the vacuolar membrane when transiently expressed in Arabidopsis protoplasts. The quantitative real-time PCR (qRT-PCR) analysis showed that GhNHX3D was induced rapidly in response to salt stress in cotton leaves, and its transcript levels increased with the aggravation of salt stress. The introduction of GhNHX3D into the salt-sensitive yeast mutant ATX3 improved its salt tolerance. Furthermore, silencing of GhNHX3D in cotton plants by virus-induced gene silencing (VIGS) increased the Na+ levels in the leaves, stems, and roots and decreased the K+ content in the roots, leading to greater salt sensitivity. Our results indicate that GhNHX3D is a member of the vacuolar NHX family and can confer salt tolerance by adjusting the steady-state balance of cellular Na+ and K+ ions.

Noncanonical Sequences Involving NHERF1 Interaction with NPT2A Govern Hormone-Regulated Phosphate Transport: Binding Outside the Box.

Na+/H+ exchange factor-1 (NHERF1), a multidomain PDZ scaffolding phosphoprotein, is required for the type II sodium-dependent phosphate cotransporter (NPT2A)-mediated renal phosphate absorption. Both PDZ1 and PDZ2 domains are involved in NPT2A-dependent phosphate uptake. Though harboring identical core-binding motifs, PDZ1 and PDZ2 play entirely different roles in hormone-regulated phosphate transport. PDZ1 is required for the interaction with the C-terminal PDZ-binding sequence of NPT2A (-TRL). Remarkably, phosphocycling at Ser290 distant from PDZ1, the penultimate step for both parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) regulation, controls the association between NHERF1 and NPT2A. PDZ2 interacts with the C-terminal PDZ-recognition motif (-TRL) of G Protein-coupled Receptor Kinase 6A (GRK6A), and that promotes phosphorylation of Ser290. The compelling biological puzzle is how PDZ1 and PDZ2 with identical GYGF core-binding motifs specifically recognize distinct binding partners. Binding determinants distinct from the canonical PDZ-ligand interactions and located "outside the box" explain PDZ domain specificity. Phosphorylation of NHERF1 by diverse kinases and associated conformational changes in NHERF1 add more complexity to PDZ-binding diversity.

The C-terminal tail of the plant endosomal-type NHXs plays a key role in its function and stability.

Typically, Na+/H+ antiporters (NHXs) possess a conserved N-terminus for cation binding and exchange and a hydrophilic C-terminus for regulating the antiporter activity. Plant endosomal-type NHXs play important roles in protein trafficking, as well as K+ and vesicle pH homeostasis, however the role of the C-terminal tail remains unclear. Here, the function of MnNHX6, an endosomal-type NHX in mulberry, was investigated using heterologous expression in yeast. Functional and localization analyses of C-terminal truncation and mutations in MnNHX6 revealed that the C-terminal conserved region was responsible for the function and stability of the protein and its hydrophobicity, which is a key domain requirement. Nuclear magnetic resonance spectroscopy provided direct structural evidence and yeast two-hybrid screening indicated that this functional domain was also necessary for interaction with sorting nexin 1. Our findings demonstrate that although the C-terminal tail of MnNHX6 is intrinsically disordered, the C-terminal conserved region may be an important part of the external mouth of this transporter, which controls protein function and stability by serving as an inter-molecular cork with a chain mechanism. These findings improve our understanding of the roles of the C-terminal tail of endosomal-type NHXs in plants and the ion transport mechanism of NHX-like antiporters.