Debajyoti Dutta

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.

Na + /H + exchanger isoform one (NHE1) is a mammalian plasma membrane protein that removes intracellular protons, thereby elevating intracellular pH (pH i). 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 trans-membrane 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. Résumé : L'échangeur Na + /H + NHE1 est une protéine de la membrane plasmique des mammifères qui enlève les protons intracellulaires, élevant ainsi le pH intracellulaire (pH i). NHE1 utilise l'énergie générée par l'entrée spontanée dans les cellules d'un sodium extracellulaire par gradient pour faire sortir un proton intracellulaire. Cette protéine ubiquiste exerce une influence importante sur les plans physiologiques et pathologiques dans les cellules de mammifères conséquemment à son activité. La structure tridimensionnelle de NHE1 humain (hNHE1) n'est pas connue. Les auteurs ont modélisé ici NHE1 à partir de la structure de MjNhaP1 de Methanocaldococcus jannaschii en combinaison avec les données biochimiques d'accessibilité de la surface. Le transporteur hNHE1 comprenait 12 segments transmembranaires dont un repli caractéristique de l'antiport Na + /H + , formé de deux segments transmembranaires dans une conformation hélice-région étendue-hélice qui se croisent à l'intérieur de la membrane. Les acides aminés 363-410 ont été cartographiés principalement à la surface extracellulaire en tant que boucle extracellulaire (EL5). Une forte prépondérance d'acides aminés accessibles à la surface selon des expériences biochimiques était cartographiée à proximité ou sur la surface extracellulaire. L'amarrage d'inhibiteurs de l'échangeur Na + /H + à la surface extra-cellulaire suggérait que la liaison de l'inhibiteur sur un site extracellulaire implique plusieurs acides aminés de différentes régions de la protéine. Les résultats présentent un nouveau modèle tridimensionnel vérifiable qui illustre la structure de NHE1 et explique les données biochimiques expérimentales. [Traduit par la Rédaction]

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 Glu 74 and Arg 77 of transmembrane segment 3. Glu 74 and Arg 77 are conserved in yeast species while Glu 74 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. Arg 341 and Arg 342 were also necessary for SpNHE1 transport in S. pombe. Deletion of 3 out of 4 acidic residues (Asp 389 , Glu 390 , Glu 392 , Glu 397) 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 coordination sphere, attracting cations for transport but that the region is not highly specific for the location of acidic charges. Overall, we identified a number of polar amino acids important in SpNHE1 function. Na + /H + exchangers are integral membrane proteins that exist in all known plants, yeast and mammalian cells 1. In mammals, they remove excess intracellular protons in exchange for external sodium and are important in ischemic heart disease and breast cancer 2,3. In contrast, in plants and yeast, plasma membrane members of this group of proteins catalyze the reverse process: exchanging intracellular sodium for extracellular protons. They therefore function in salt tolerance extruding sodium ions through these plasma membrane transporters. The energy of transport comes from the proton gradient generated by the plasma membrane H +-ATPase 4. In plants, salt tolerance is significant in agriculture and in this regard, overexpression of plasma membrane salt tolerance proteins has been shown to improve salt tolerance in plants 5. In the fission yeast Schizosaccharomyces pombe, the Na + /H + exchanger SpNHE1 (previously known as sod2) plays the major role in salt removal from the cytosol and in salt tolerance. It is an excellent model system with which to study plasma membrane salt tolerance proteins 6. S. pombe has limited other salt tolerance mechanisms in the plasma membrane, so the deletion of SpNHE1 causes this yeast to exhibit a salt sensitive phenotype 7. By reintroducing the protein, salt tolerance is restored and this allows assay of defective protein's activity. We have used this assay in earlier works 6,8,9. SpNHE1 can also enhance salt tolerance in plants 10 and in phylogenetic analysis SpNHE1 clusters with plant plasma membrane salt tolerance proteins 11. The mechanism of transport of plasma membrane salt tolerance proteins and Na + /H + exchangers in general, is of interest both as a fundamental scientific problem and from the economic view of modification of plants to improve agricultural output. Crystal structures of some forms of Na + /H + exchanger are known such the E. coli sodium transporter NhaA 12 , Thermos thermophilus (NapA) 13 , Pyrococcus abyssi (PaNhaP) 14 and Methanococcus

In Mycobacterium tuberculosis, acyl carrier protein (AcpM)-mediated fatty
acid synthase type II is integral for the synthesis of mycolic acids. AcpM,
designated as an atypical ACP, comprises of a putative 33 amino acid long
C-terminal extension which is distinctive in nature. Here, we aimed at devising
an ‘easy-to-go’ method for the generation of crypto-AcpM loaded with a
solvatochromic probe 7-Nitrobenz-2-oxa-1,3-diazol-4-yl, which is linked to
the 40-phosphopantetheine (Ppant) prosthetic group of AcpM. The crypto-
AcpM, coupled with fluorescence spectroscopy and molecular dynamics simulation studies, was employed to explore the elusive dynamics of Ppant arm in AcpM. This investigation establishes the role of the flexible C-terminal extension of AcpM in regulating the prosthetic group sequestration ability by modulating the ‘Asp-Ser-Leu’ motif.

The mammalian Na+/H+ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pHi) by removing a single intracellular proton in exchange for one extracellular sodium ion. It is involved in cardiac hypertrophy and ischemia reperfusion damage to the heart and elevation of its activity is a trigger for breast cancer metastasis. NHE1 has an extensive 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments connected by intracellular and extracellular loops. Intracellular loops are hypothesized to modulate the sensitivity to pHi. In this study, we characterized the structure and function of intracellular loop 5 (IL5), specifically amino acids 431-443. Mutation of eleven residues to alanine caused partial or nearly complete inhibition of transport; notably, mutation of residues L432, T433, I436, N437, R440 and K443 demonstrated these residues had critical roles in NHE1 function independent of effects on targeting or expression. The nuclear magnetic resonance (NMR) solution spectra of the IL5 peptide in a membrane mimetic sodium dodecyl sulfate solution revealed that IL5 has a stable three-dimensional structure with substantial alpha helical character. NMR chemical shifts indicated that K438 was in close proximity with W434. Overall, our results show that IL5 is a critical, intracellular loop with a propensity to form an alpha helix, and many residues of this intracellular loop are critical to proton sensing and ion transport.

FabG belongs to the short-chain dehydrogenase/reductase (SDR) family. As a part of fatty acid elongation cycle, the
enzyme executes the 2nd step of the cycle by reducing β-ketoacyl-acyl carrier protein using NADPH cofactor. The insolution
oligomeric organisations of FabGs tend to differ in various organisms although the crystal structures are
mainly tetrameric. Importantly, this oligomerization propensity is crucial because it directs enzymatic activation upon
cofactor binding and substrate cooperativity. Here, the aim of our work is to find out the underlying molecular basis that
switches the different oligomeric conformations of FabGs as well as how it is associated with the presence of cofactor
and substrate. To establish our proposition a chimeric FabG has been designed by using domain swapping strategy.
Our data suggest that the substitution of a modified P-interface can alter the dimer-tetramer equilibrium in FabGs.
Overall, the study provides insights into the structural dynamics of FabGs and allows us to understand the key
structural features that regulate oligomeric conformation as well as the substrate, and cofactor binding.

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 Glu361 may be involved in cation coordination on the cytoplasmic face of the protein with a negative charge in this position being important. His367, Ile371 and Gly372 were important in function. Ile371 may have important hydrophobic interactions with other residues and Gly372 may be important in maintaining an extended conformation. Several residues from Val377 to Leu384 are important in function possibly involved in hydrophobic interactions with other amino acids. We suggest that TM XI forms part of the ion translocation core of this Na+/H+ exchanger.

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 R-S-Q-X-E 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 and it represents the first structure of a DNA-targeting mono-ADP-ribosyltransferase enzyme; the structures of the apo form (1.45Å) and with two inhibitors (P6-E, 1.4Å; PJ34, 1.6Å) were solved. These structures are also the first high-resolution models of the Pierisin subgroup of the mono-ADP-ribosyltransferase toxin family. A model of Scabin with its DNA substrate is also proposed.

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

C3larvin toxin is a new member of the C3 class of the mono-ADP-ribosyltransferase (mART) toxin family. The C3 toxins are known to covalently modify small G-proteins, e.g. RhoA, impairing their function, and serving as virulence factors for an offending pathogen. A full-length X-ray structure of C3larvin (2.3 Å) revealed that the characteristic mixed α/β-fold consists of a central β-core flanked by two helical regions. Topologically, the protein can be separated into N and C lobes, each formed by a β-sheet and an α-motif and connected by exposed loops involved in the recognition, binding and catalysis of the toxin/enzyme i.e., the ADP-ribosylation turn-turn (ARTT) and phosphate-nicotinamide PN loops. Herein, we provide two new C3larvin X-ray structures and present a systematic study of the toxin dynamics by first analysing the experimental variability of the X-ray dataset followed by contrasting those results with theoretical predictions based on Elastic Network Models (GNM and ANM)....

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.