Ph. D in protein crystallography on Mycobacterium tuberculosis fatty acid metabolism proteins from Indian Institute of Technology Kharagpur, pre-Ph.D in Harvard Medical School, Postdoctoral fellow in the University of Guelph on bacterial ADP-ribosylating toxins and toxin-target complexes, Post doctoral fellow in University of Alberta on Yeast and Plant sodium proton antiporters.
Context Calcium-dependent signaling in plants is responsible for several major cellular events, i... more 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-Ca 2+ 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 Ca 2+-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.
High salinity in agricultural lands is one of the predominant issues limiting agricultural yields... more 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.
Plants have several mechanisms to endure salinity stress. The degree of salt tolerance varies sig... more 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.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2021
Mammalian Na+/H+ exchanger isoform one (NHE1) is a plasma membrane protein responsible for pH reg... more 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.
Isoform one of the mammalian Na+/H+ exchanger is a plasma membrane protein that is ubiquitously p... more 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, ab...
The Na+/H+ exchanger of the plasma membrane of S. pombe (SpNHE1) removes excess intracellular sod... more The Na+/H+ exchanger of the plasma membrane of S. pombe (SpNHE1) removes excess intracellular sodium in exchange for an extracellular proton. We examined the functional role of acidic amino acids of a yeast specific periplasmic extracellular loop 6 (EL6) and of Glu74 and Arg77 of transmembrane segment 3. Glu74 and Arg77 are conserved in yeast species while Glu74 is conserved throughout various phyla. The mutation E74A caused a minor effect, while mutation R77A had a larger effect on the ability of SpNHE1 to confer salt tolerance. Mutation of both residues to Ala or Glu also eliminated the ability to confer salt tolerance. Arg341 and Arg342 were also necessary for SpNHE1 transport in S. pombe. Deletion of 3 out of 4 acidic residues (Asp389, Glu390, Glu392, Glu397) of EL6 did not greatly affect SpNHE1 function while deletion of all did. Replacement of EL6 with a segment from the plant Na+/H+ exchanger SOS1 also did not affect function. We suggest that EL6 forms part of a cation coordi...
Sodium proton antiporters (or sodium proton exchangers [NHEs]) are a critical family of membrane ... more 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 thes...
The Na(+)/H(+) exchanger of the plasma membrane of S. pombe (SpNHE1) removes intracellular sodium... more The Na(+)/H(+) exchanger of the plasma membrane of S. pombe (SpNHE1) removes intracellular sodium in exchange for an extracellular proton. We examined the structure and functional role of amino acids 360-393 of putative transmembrane (TM) segment XI of SpNHE1. Structural analysis suggested that it had a helical propensity over amino acids 360-368, an extended region from 369-378 and was helical over amino acids 379-386. TM XI was sensitive to side chain alterations. Mutation of eight amino acids to alanine resulted in loss of one or both of LiCl or NaCl tolerance when re-introduced into SpNHE1 deficient S. pombe. Mutation of seven other amino acids had minor effects. Analysis of structure and functional mutations suggested that Glu(361) may be involved in cation coordination on the cytoplasmic face of the protein with a negative charge in this position being important. His(367), Ile(371) and Gly(372) were important in function. Ile(371) may have important hydrophobic interactions wi...
Context Calcium-dependent signaling in plants is responsible for several major cellular events, i... more 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-Ca 2+ 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 Ca 2+-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.
High salinity in agricultural lands is one of the predominant issues limiting agricultural yields... more 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.
Plants have several mechanisms to endure salinity stress. The degree of salt tolerance varies sig... more 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.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2021
Mammalian Na+/H+ exchanger isoform one (NHE1) is a plasma membrane protein responsible for pH reg... more 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.
Isoform one of the mammalian Na+/H+ exchanger is a plasma membrane protein that is ubiquitously p... more 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, ab...
The Na+/H+ exchanger of the plasma membrane of S. pombe (SpNHE1) removes excess intracellular sod... more The Na+/H+ exchanger of the plasma membrane of S. pombe (SpNHE1) removes excess intracellular sodium in exchange for an extracellular proton. We examined the functional role of acidic amino acids of a yeast specific periplasmic extracellular loop 6 (EL6) and of Glu74 and Arg77 of transmembrane segment 3. Glu74 and Arg77 are conserved in yeast species while Glu74 is conserved throughout various phyla. The mutation E74A caused a minor effect, while mutation R77A had a larger effect on the ability of SpNHE1 to confer salt tolerance. Mutation of both residues to Ala or Glu also eliminated the ability to confer salt tolerance. Arg341 and Arg342 were also necessary for SpNHE1 transport in S. pombe. Deletion of 3 out of 4 acidic residues (Asp389, Glu390, Glu392, Glu397) of EL6 did not greatly affect SpNHE1 function while deletion of all did. Replacement of EL6 with a segment from the plant Na+/H+ exchanger SOS1 also did not affect function. We suggest that EL6 forms part of a cation coordi...
Sodium proton antiporters (or sodium proton exchangers [NHEs]) are a critical family of membrane ... more 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 thes...
The Na(+)/H(+) exchanger of the plasma membrane of S. pombe (SpNHE1) removes intracellular sodium... more The Na(+)/H(+) exchanger of the plasma membrane of S. pombe (SpNHE1) removes intracellular sodium in exchange for an extracellular proton. We examined the structure and functional role of amino acids 360-393 of putative transmembrane (TM) segment XI of SpNHE1. Structural analysis suggested that it had a helical propensity over amino acids 360-368, an extended region from 369-378 and was helical over amino acids 379-386. TM XI was sensitive to side chain alterations. Mutation of eight amino acids to alanine resulted in loss of one or both of LiCl or NaCl tolerance when re-introduced into SpNHE1 deficient S. pombe. Mutation of seven other amino acids had minor effects. Analysis of structure and functional mutations suggested that Glu(361) may be involved in cation coordination on the cytoplasmic face of the protein with a negative charge in this position being important. His(367), Ile(371) and Gly(372) were important in function. Ile(371) may have important hydrophobic interactions wi...
High salt in agricultural lands or coastal areas is one of the prevalent problems in agriculture.... more High salt in agricultural lands or coastal areas is one of the prevalent problems in agriculture. It limits agricultural productivity because most crops cannot grow well in the presence of high salt concentrations. However, every plant can tolerate salinity to some extent. The salt tolerance pathways involving membrane proteins play a crucial role in withstanding the salinity stress. These membrane proteins consist of ion transporters, signaling receptors, signaling molecule generators, and solute transporters. Salt tolerance in the plant can be improved by modulating membrane proteins' function, distribution, or expression. CRISPR technology provides an unprecedented opportunity to attain this modification of the plant genome with several advantages. The process is easy, less time-intensive, and highly efficient. This chapter will highlight the functions of plant membrane proteins in salt tolerance and discuss their importance as CRISPR targets. Additionally, the chapter will present up-to-date knowledge and application of CRISPR technology in plants to modulate protein's function and expression in salt tolerance.
High salt in agricultural lands or coastal areas is one of the prevalent problems in agriculture.... more High salt in agricultural lands or coastal areas is one of the prevalent problems in agriculture. It limits agricultural productivity because most crops cannot grow well in the presence of high salt concentrations. However, every plant can tolerate salinity to some extent. The salt tolerance pathways involving membrane proteins play a crucial role in withstanding the salinity stress. These membrane proteins consist of ion transporters, signaling receptors, signaling molecule generators, and solute transporters. Salt tolerance in the plant can be improved by modulating membrane proteins' function, distribution, or expression. CRISPR technology provides an unprecedented opportunity to attain this modification of the plant genome with several advantages. The process is easy, less time-intensive, and highly efficient. This chapter will highlight the functions of plant membrane proteins in salt tolerance and discuss their importance as CRISPR targets. Additionally, the chapter will present up-to-date knowledge and application of CRISPR technology in plants to modulate protein's function and expression in salt tolerance.
High salt in agricultural lands or coastal areas is one of the prevalent problems in agriculture.... more High salt in agricultural lands or coastal areas is one of the prevalent problems in agriculture. It limits agricultural productivity because most crops cannot grow well in the presence of high salt concentrations. However, every plant can tolerate salinity to some extent. The salt tolerance pathways involving membrane proteins play a crucial role in withstanding the salinity stress. These membrane proteins consist of ion transporters, signaling receptors, signaling molecule generators, and solute transporters. Salt tolerance in the plant can be improved by modulating membrane proteins' function, distribution, or expression. CRISPR technology provides an unprecedented opportunity to attain this modification of the plant genome with several advantages. The process is easy, less time-intensive, and highly efficient. This chapter will highlight the functions of plant membrane proteins in salt tolerance and discuss their importance as CRISPR targets. Additionally, the chapter will present up-to-date knowledge and application of CRISPR technology in plants to modulate protein's function and expression in salt tolerance.
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Papers by Debajyoti Dutta