Membrane Proteins in Plant Salinity Stress Perception, Sensing, and Response.

Plants have several mechanisms to endure salinity stress. The degree of salt tolerance varies significantly among different terrestrial crops. Proteins at the plant's cell wall and membrane mediate different physiological roles owing to their critical positioning between two distinct environments. A specific membrane protein is responsible for a single type of activity, such as a specific group of ion transport or a similar group of small molecule binding to exert multiple cellular effects. During salinity stress in plants, membrane protein functions: ion homeostasis, signal transduction, redox homeostasis, and solute transport are essential for stress perception, signaling, and recovery. Therefore, comprehensive knowledge about plant membrane proteins is essential to modulate crop salinity tolerance. This review gives a detailed overview of the membrane proteins involved in plant salinity stress highlighting the recent findings. Also, it discusses the role of solute transporters, accessory polypeptides, and proteins in salinity tolerance. Finally, some aspects of membrane proteins are discussed with potential applications to developing salt tolerance in crops.

Interplay between membrane proteins and membrane protein-lipid pertaining to plant salinity stress.

High salinity in agricultural lands is one of the predominant issues limiting agricultural yields. Plants have developed several mechanisms to withstand salinity stress, but the mechanisms are not effective enough for most crops to prevent and persist the salinity stress. Plant salt tolerance pathways involve membrane proteins that have a crucial role in sensing and mitigating salinity stress. Due to a strategic location interfacing two distinct cellular environments, membrane proteins can be considered checkpoints to the salt tolerance pathways in plants. Related membrane proteins functions include ion homeostasis, osmosensing or ion sensing, signal transduction, redox homeostasis, and small molecule transport. Therefore, modulating plant membrane proteins' function, expression, and distribution can improve plant salt tolerance. This review discusses the membrane protein-protein and protein-lipid interactions related to plant salinity stress. It will also highlight the finding of membrane protein-lipid interactions from the context of recent structural evidence. Finally, the importance of membrane protein-protein and protein-lipid interaction is discussed, and a future perspective on studying the membrane protein-protein and protein-lipid interactions to develop strategies for improving salinity tolerance is proposed.

Rainbow Trout (Oncorhynchus mykiss) Na+/H+ Exchangers tNhe3a and tNhe3b Display Unique Inhibitory Profiles Dissimilar from Mammalian NHE Isoforms.

Freshwater fishes maintain an internal osmolality of ~300 mOsm, while living in dilute environments ranging from 0 to 50 mOsm. This osmotic challenge is met at least partially, by Na+/H+ exchangers (NHE) of fish gill and kidney. In this study, we cloned, expressed, and pharmacologically characterized fish-specific Nhes of the commercially important species Oncorhynchus mykiss. Trout (t) Nhe3a and Nhe3b isoforms from gill and kidney were expressed and characterized in an NHE-deficient cell line. Western blotting and immunocytochemistry confirmed stable expression of the tagged trout tNhe proteins. To measure NHE activity, a transient acid load was induced in trout tNhe expressing cells and intracellular pH was measured. Both isoforms demonstrated significant activity and recovered from an acute acid load. The effect of the NHE transport inhibitors amiloride, EIPA (5-(N-ethyl-N-isopropyl)-amiloride), phenamil, and DAPI was examined. tNhe3a was inhibited in a dose-dependent manner by amiloride and EIPA and tNhe3a was more sensitive to amiloride than EIPA, unlike mammalian NHE1. tNhe3b was inhibited by high concentrations of amiloride, while even in the presence of high concentrations of EIPA (500 µM), some activity of tNhe3b remained. Phenamil and DAPI were ineffective at inhibiting tNhe activity of either isoform. The current study aids in understanding the pharmacology of fish ion transporters. Both isoforms display inhibitory profiles uniquely different from mammalian NHEs and show resistance to inhibition. Our study allows for more direct interpretation of past, present, and future fish-specific sodium transport studies, with less reliance on mammalian NHE data for interpretation.

Unprecedented Properties of Phenothiazines Unraveled by a NDH-2 Bioelectrochemical Assay Platform.

Type II NADH:quinone oxidoreductase (NDH-2) plays a crucial role in the respiratory chains of many organisms. Its absence in mammalian cells makes NDH-2 an attractive new target for developing antimicrobials and antiprotozoal agents. We established a novel bioelectrochemical platform to characterize the catalytic behavior of NDH-2 from Caldalkalibacillus thermarum and Listeria monocytogenes strain EGD-e while bound to native-like lipid membranes. Catalysis of both NADH oxidation and lipophilic quinone reduction by membrane-bound NDH-2 followed the Michaelis-Menten model; however, the maximum turnover was only achieved when a high concentration of quinone (>3 mM) was present in the membrane, suggesting that quinone availability regulates NADH-coupled respiration activity. The quinone analogue 2-heptyl-4-hydroxyquinoline-N-oxide inhibited C. thermarum NDH-2 activity, and its potency is higher in a membrane environment compared to assays performed with water-soluble quinone analogues, demonstrating the importance of testing compounds under physiologically relevant conditions. Furthermore, when phenothiazines, one of the most commonly identified NDH-2 inhibitors, were tested, they did not inhibit membrane-bound NDH-2. Instead, our assay platform unexpectedly suggests a novel mode of phenothiazine action where chlorpromazine, a promising antitubercular agent and key medicine used to treat psychotic disorders, is able to disrupt pH gradients across bacterial membranes.

Amino Acids 563-566 of the Na+/H+ Exchanger Isoform 1 C-Terminal Cytosolic Tail Prevent Protein Degradation and Stabilize Protein Expression and Activity.

Isoform one of the mammalian Na+/H+ exchanger is a plasma membrane protein that is ubiquitously present in humans. It regulates intracellular pH through the removal of one intracellular proton in exchange for a single extracellular sodium. It consists of a 500 amino acid membrane domain plus a 315 amino acid, C-terminal tail. We examined amino acids of the C-terminal tail that are important in the targeting and activity of the protein. A previous study demonstrated that stop codon polymorphisms can result in decreased activity, expression, targeting and enhanced protein degradation. Here, we determine elements that are critical in these anomalies. A series of progressive deletions of the C-terminal tail demonstrated a progressive decrease in activity and targeting, though these remained until a final drop off with the deletion of amino acids 563-566. The deletion of the 562LIAGERS568 sequence or the alteration to the 562LAAAARS568 sequence caused the decreased protein expression, aberrant targeting, reduced activity and enhanced degradation of the Na+/H+ exchanger (NHE1) protein. The 562LIAGERS568 sequence bound to other regions of the C-terminal cytosolic domain. We suggest this region is necessary for the activity, targeting, stability, and expression of the NHE1 protein. The results define a new sequence that is important in maintenance of NHE1 protein levels and activity.

Molecular modeling and inhibitor docking analysis of the Na+/H+ exchanger isoform one 1.

Na+/H+ exchanger isoform one (NHE1) is a mammalian plasma membrane protein that removes intracellular protons, thereby elevating intracellular pH (pHi). NHE1 uses the energy of allowing an extracellular sodium down its gradient into cells to remove one intracellular proton. The ubiquitous protein has several important physiological and pathological influences on mammalian cells as a result of its activity. The three-dimensional structure of human NHE1 (hNHE1) is not known. Here, we modeled NHE1 based on the structure of MjNhaP1 of Methanocaldoccocus jannaschii in combination with biochemical surface accessibility data. hNHE1 contained 12 transmembrane segments including a characteristic Na+/H+ antiporter fold of two transmembrane segments with a helix - extended region - helix conformation crossing each other within the membrane. Amino acids 363-410 mapped principally to the extracellular surface as an extracellular loop (EL5). A large preponderance of amino acids shown to be surface accessible by biochemical experiments mapped near to, or on, the extracellular surface. Docking of Na+/H+ exchanger inhibitors to the extracellular surface suggested that inhibitor binding on an extracellular site is made up from several amino acids of different regions of the protein. The results present a novel testable, three-dimensional model illustrating NHE1 structure and accounting for experimental biochemical data.

Structure and function of yeast and fungal Na+ /H+ antiporters.

Sodium proton antiporters (or sodium proton exchangers [NHEs]) are a critical family of membrane proteins that exchange sodium for protons across cell membranes. In yeast and plants, their primary function is to keep the sodium concentration low inside the cytoplasm. One class of NHE constitutively expressed in yeast is the plasma membrane Na+ /H+ antiporter, and another class is expressed on the endosomal/vacuolar membrane. At present, four bacterial plasma membrane antiporter structures are known and nuclear magnetic resonance structures are available for the membrane spanning transmembrane helices of mammalian and yeast NHEs. Additionally, a vast amount of mutational data are available on the role of individual amino acids and critical motifs involved in transport. We combine this information to obtain a more detailed picture of the yeast NHE plasma membrane protein and review mechanisms of transport, conserved motifs, unique residues important in function, and regulation of these proteins. The Na+ /H+ antiporter of Schizosaccharomyces pombe, SpNHE1, is an interesting model protein in an easy to study system and is representative of fungal Na+ /H+ antiporters. © IUBMB Life, 70(1):23-31, 2018.

Scabin, a Novel DNA-acting ADP-ribosyltransferase from Streptomyces scabies.

A bioinformatics strategy was used to identify Scabin, a novel DNA-targeting enzyme from the plant pathogen 87.22 strain of Streptomyces scabies Scabin shares nearly 40% sequence identity with the Pierisin family of mono-ADP-ribosyltransferase toxins. Scabin was purified to homogeneity as a 22-kDa single-domain enzyme and was shown to possess high NAD(+)-glycohydrolase (Km (NAD) = 68 ± 3 µm; kcat = 94 ± 2 min(-1)) activity with an RSQXE motif; it was also shown to target deoxyguanosine and showed sigmoidal enzyme kinetics (K0.5(deoxyguanosine) = 302 ± 12 µm; kcat = 14 min(-1)). Mass spectrometry analysis revealed that Scabin labels the exocyclic amino group on guanine bases in either single-stranded or double-stranded DNA. Several small molecule inhibitors were identified, and the most potent compounds were found to inhibit the enzyme activity with Ki values ranging from 3 to 24 µm PJ34, a well known inhibitor of poly-ADP-ribosyltransferases, was shown to be the most potent inhibitor of Scabin. Scabin was crystallized, representing the first structure of a DNA-targeting mono-ADP-ribosyltransferase enzyme; the structures of the apo-form (1.45 Å) and with two inhibitors (P6-E, 1.4 Å; PJ34, 1.6 Å) were solved. These x-ray structures are also the first high resolution structures of the Pierisin subgroup of the mono-ADP-ribosyltransferase toxin family. A model of Scabin with its DNA substrate is also proposed.

Expression and characterization of the SOS1 Arabidopsis salt tolerance protein.

SOS1 is the plasma membrane Na(+)/H(+) antiporter of Arabidopsis thaliana. It is responsible for the removal of intracellular sodium in exchange for an extracellular proton. SOS1 is composed of 1146 amino acids. Approximately 450 make the membrane domain, while the protein contains and a very large regulatory cytosolic domain of about 696 amino acids. Schizosaccharomyces pombe contains the salt tolerance Na(+)/H(+) antiporter proteins sod2. We examined the ability of SOS1 to rescue salt tolerance in S. pombe with a knockout of the sod2 gene (sod2::ura4). In addition, we characterized the importance of the regulatory tail of SOS1, in expression of the protein in S. pombe. We expressed full-length SOS1 and SOS1 shortened at the C-terminus and ending at amino acids 766 (medium) and 481 (short). The short version of SOS1 conveyed salt tolerance to sod2::ura4 yeast and Western blotting revealed that the protein was present. The protein was also targeted to the plasma membrane. The medium and full-length SOS1 protein were partially degraded and were not as well expressed as the short version of SOS1. The SOS1 short protein was also able to reduce Na(+) content in S. pombe. The full-length SOS1 dimerized and depended on the presence of the cytosolic tail. An analysis of SOS1 predicted a topology of 13 transmembrane segments, distinct from E. coli NhaA but similar to the Na(+)/H(+) exchangers Methanocaldococcus jannaschii NhaP1 and Thermus thermophile NapA.

Crystal structure of dehydratase component HadAB complex of mycobacterial FAS-II pathway.

Fatty acid biosynthesis type II in mycobacteria delivers the fatty acids required for mycolic acid synthesis. The pathway employs a unique maoC like ß-hydroxyacyl-ACP dehydratase HadAB or HadBC heterodimer in the third step of the elongation cycle. Here we report the crystal structure of the HadAB complex determined using a Pb-SIRAS method. Crystal structure aided with enzymatic study establishes the roles of HadA as a scaffolding component and HadB as a catalytic component together indispensable for the activity. The detailed structural analysis of HadAB in combination with MD simulation endorses the spatial orientation of the central hot-dog helix and the dynamic nature of its associated loop in regulation of substrate specificities in dehydratase/hydratase family enzymes.

Design, synthesis and characterization of novel inhibitors against mycobacterial ß-ketoacyl CoA reductase FabG4.

We report the design and synthesis of triazole-polyphenol hybrid compounds 1 and 2 as inhibitors of the FabG4 (Rv0242c) enzyme of Mycobacterium tuberculosis for the first time. A major advance in this field occurred only a couple of years ago with the X-ray crystal structure of FabG4, which has helped us to design these inhibitors by the computational fragment-based drug design (FBDD) approach. Compound 1 has shown competitive inhibition with an inhibition constant (Ki) value of 3.97 ± 0.02 µM. On the other hand, compound 2 has been found to be a mixed type inhibitor with a Ki value of 0.88 ± 0.01 µM. Thermodynamic analysis using isothermal titration calorimetry (ITC) reveals that both inhibitors bind at the NADH co-factor binding domain. Their MIC values, as determined by resazurin assay against M. smegmatis, indicated their good anti-mycobacterial properties. A preliminary structure-activity relationship (SAR) study supports the design of these inhibitors. These compounds may be possible candidates as lead compounds for alternate anti-tubercular drugs. All of the reductase enzymes of the Mycobacterium family have a similar ketoacyl reductase (KAR) domain. Hence, this work may be extrapolated to find structure-based inhibitors of other reductase enzymes.

Crystal structure of hexanoyl-CoA bound to ß-ketoacyl reductase FabG4 of Mycobacterium tuberculosis.

FabGs, or ß-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of ß-oxoacyl-ACP (acyl-carrier protein) into ß-hydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal 'flavodoxintype' domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce ß-oxoacyl-CoA to ß-hydroxyacyl-CoA. In the present study the crystal structures of the FabG4-NADH binary complex and the FabG4-NAD+-hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within the active site cavity of FabG significantly differs from that of the C16 fattyacyl substrate bound to mycobacterial FabI [InhA (enoyl-ACP reductase)]. The ternary complex reveals that both loop I and loop II interact with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural data ACP inhibition studies indicate that FabG4 can accept both CoA- and ACP-based fattyacyl substrates. We have also shown that in the FabG4 dimer Arg146 and Arg445 of one monomer interact with the C-terminus of the second monomer to play pivotal role in substrate association and catalysis.

Structural and molecular dynamics simulation studies of CBL-interacting protein kinase CIPK and its complexes related to plant salinity stress.

CONTEXT: Calcium-dependent signaling in plants is responsible for several major cellular events, including the activation of the salinity-responsive pathways. Calcium binds to calcineurin B-like protein (CBL), and the resulting CBL-Ca2+ complex binds to CBL-interacting protein kinase (CIPK). The CBL-CIPK complex enhances the CIPK interaction with an upstream kinase. The upstream kinase phosphorylates CIPK that, in turn, phosphorylates membrane transporters. Phosphorylation influences transporter activity to kick-start many downstream functions, such as balancing the cytosolic Na+-to-K+ ratio. The CBL-CIPK interaction is pivotal for Ca2+-dependent salinity stress signaling. METHODS: Computational methods are used to model the entire Arabidopsis thaliana CIPK24 protein structure in its autoinhibited and open-activated states. Arabidopsis thaliana CIPK24-CBL4 complex is predicted based on the protein-protein docking methods. The available structural and functional data support the CIPK24 and the CIPK24-CBL4 complex models. Models are energy-minimized and subjected to molecular dynamics (MD) simulations. MD simulations for 500 ns and 300 ns enabled us to predict the importance of conserved residues of the proteins. Finally, the work is extended to predict the CIPK24-CBL4 complex with the upstream kinase GRIK2. MD simulation for 300 ns on the ternary complex structure enabled us to identify the critical CIPK24-GRIK2 interactions. Together, these data could be used to engineer the CBL-CIPK interaction network for developing salt tolerance in crops.

Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of Rv0241c (HtdX) from Mycobacterium tuberculosis H37Rv.

Rv0241c (HtdX) is a putative (3R)-hydroxyacyl-CoA dehydratase of Mycobacterium tuberculosis. The htdX gene belongs to a conserved operon and is expressed in mycobacteria in the presence of several fatty-acid synthase II drugs. To elucidate the structure of HtdX, the protein was cloned, overexpressed, purified to homogeneity and crystallized. The protein was crystallized from two conditions: (i) 3 M sodium chloride, 0.1 M Na HEPES pH 8.0 and (ii) 2.5 M sodium chloride, 0.1 M Tris-HCl pH 8.5. A complete diffraction data set was collected from crystals from both conditions. The crystal from the first condition diffracted to 2.3 Šresolution and belonged to space group I41, with unit-cell parameters a=b=61.51, c=143.81 Å. Crystals from the second condition diffracted to 3.1 Šresolution and belonged to space group P43212 or P41212, with unit-cell parameters a=b=63.67, c=140.88 Å. Both crystals contained one molecule in the asymmetric unit.

Crystallization and preliminary X-ray diffraction analysis of the high molecular weight ketoacyl reductase FabG4 complexed with NADH.

FabG4 from Mycobacterium tuberculosis belongs to the high molecular weight ketoacyl reductases (HMwFabGs). The enzyme requires NADH for ß-ketoacyl reductase activity. The protein was overexpressed, purified to homogeneity and crystallized as a FabG4-NADH complex. A mountable FabG4:NADH complex crystal diffracted to 2.59 Šresolution and belonged to space group P1, with unit-cell parameters a = 63.07, b = 71.03, c = 92.92 Å, α = 105.02, ß = 97.06, γ = 93.66°. The Matthews coefficient suggested the presence of four monomers in the unit cell. In addition, a self-rotation function revealed the presence of two twofold NCS axes and one fourfold NCS axis. At χ = 180° the highest peak corresponds to the twofold NCS between two monomers, whereas the second peak corresponds to the twofold NCS between two dimers.

Crystal structure of FabG4 from Mycobacterium tuberculosis reveals the importance of C-terminal residues in ketoreductase activity.

Rv0242c, also known as FabG4, is a beta-ketoacyl CoA reductase in Mycobacterium tuberculosis. The crystal structure of C-terminal truncated FabG4 is solved at 2.5Å resolution which shows the presence of two distinct domains, domain I and II. Domain I partially resembles "flavodoxin type domain" and the domain II is a typical "ketoacyl CoA reductase (KAR) domain". The enzyme exhibits ketoacyl CoA reductase activity by reducing acetoacyl CoA to 3-hydroxyacyl CoA in presence of NADH. Conserved catalytic triad Ser347, Tyr360, and Lys364 constitute the active site residues of the KAR domain. Presence of the Tyr and the Lys residues in the triad in a particular orientation is imperative for effective catalytic mechanism. The importance of loop I and II and the role of the C-terminal residues of KAR domain are highlighted. Comparative structural analyses clearly demonstrate that loop II is stabilized by hydrophobic interaction with C-terminal residues to sustain the orientation of Tyr360. Loop I interacts with loop II via H-bonding network to restrict the active site residue Lys364 in a catalytically favorable orientation.

Design, synthesis and inhibition activity of a novel cyclic enediyne amino acid conjugates against MPtpA.

In course of studies towards the discovery of selective inhibitors of MPtpA, a novel cyclic endiyne malonamic acid has been designed and synthesized. The synthesis involves a crucial intramolecular Knoevenagel reaction. The compound displayed a reversible non-competitive inhibition against MPtpA with inhibition constant K(i) of 22.5 µM. The enediyne acts as a recognition framework in inducing the inhibition and not as a reactive functional moiety. This was confirmed by comparing the inhibiting activity with that of the corresponding saturated cyclic non-enediyne analogue.

Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) from Staphylococcus aureus MSSA476.

The gene product of the sas2203 ORF of Staphylococcus aureus MSSA476 encodes a 30 kDa molecular-weight protein with a high sequence resemblance (29% identity) to tetrameric inositol monophosphatase from Thermotoga maritima. The protein was cloned, expressed, purified to homogeneity and crystallized. Crystals appeared in several conditions and good diffraction-quality crystals were obtained from 0.2 M Li(2)SO(4), 20% PEG 3350, 0.1 M HEPES pH 7.0 using the sitting-drop vapour-diffusion method. A complete diffraction data set was collected to 2.6 Šresolution using a Rigaku MicroMax-007 HF Cu Kα X-ray generator and a Rigaku R-AXIS IV(++) detector. The diffraction data were consistent with the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 49.98, b = 68.35, c = 143.79 Å, α = ß = γ = 90°, and the crystal contained two molecules in the asymmetric unit.

Characterization of modeled inhibitory binding sites on isoform one of the Na+/H+ exchanger.

Mammalian Na+/H+ exchanger isoform one (NHE1) is a plasma membrane protein responsible for pH regulation in mammalian cells. Excess activity of the protein promotes heart disease and is a trigger of metastasis in cancer. Inhibitors of the protein exist but problems in specificity have delayed their clinical application. Here we examined amino acids involved in two modeled inhibitor binding sites (A, B) in human NHE1. Twelve mutations (Asp159, Phe348, Ser351, Tyr381, Phe413, Leu465, Gly466, Tyr467, Leu468, His473, Met476, Leu481) were made and characterized. Mutants S351A, F413A, Y467A, L468A, M476A and L481A had 40-70% of wild type expression levels, while G466A and H473A expressed 22% ~ 30% of the wild type levels. Most mutants, were targeted to the cell surface at levels similar to wild type NHE1, approximately 50-70%, except for F413A and G466A, which had very low surface targeting. Most of the mutants had measurable activity except for D159A, F413A and G466A. Resistance to inhibition by EMD87580 was elevated in mutants F438A, L465A and L468A and reduced in mutants S351A, Y381A, H473A, M476A and L481A. All mutants with large alterations in inhibitory properties showed reduced Na+ affinity. The greatest changes in activity and inhibitor sensitivity were in mutants present in binding site B which is more closely associated with TM4 and C terminal of extracellular loop 5, and is situated between the putative scaffolding domain and transport domain. The results help define the inhibitor binding domain of the NHE1 protein and identify new amino acids involved in inhibitor binding.

Design, synthesis and inhibition activity of novel cyclic peptides against protein tyrosine phosphatase A from Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent for tuberculosis has employed several signalling molecules to sense the host cellular environment and act accordingly. For example, protein tyrosine phosphatase A (MPtpA) of M. tuberculosis, a signalling protein belonging to the tyrosine phosphatase superfamily, is involved in phagocytosis and is active in virulent mycobacterial form. Starting from a ß-lactam framework a new class of structure based cyclic peptide (CP) inhibitors was designed. The synthesis involves a crucial intramolecular transamidation via a ring opening reaction. All the compounds show moderate to good inhibitory activities against MPtpA in micromolar concentrations. The results of inhibition kinetics suggest mixed mode of inhibition. The binding constant determined from circular dichroism (CD) and fluorescence quenching studies shows strong binding of the hydrophilic side chain of CPs with the enzyme active site residues. All these are well supported by docking studies.