Rajan Sankaranarayanan

Centre for Cellular and Molecular Biology, Bhaganagar, Andhra Pradesh, India

Are you Rajan Sankaranarayanan?

Claim your profile

Publications (61)330.22 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Substrate binding to enzymes often follows a precise order where catalysis is accomplished through programmed conformational changes. Short-chain dehydrogenase/reductase (SDR) enzymes follow sequential order ‘bi–bi’ reaction kinetics. The mechanistic study of a SDR homolog, reductase (R) domain, from multifunctional enzymes, e.g. Nonribosomal Peptide Synthetases (NRPSs) and Polyketide Synthases (PKSs) has revealed that it reductively releases 4′-phosphopantetheinyl arm-tethered peptidyl product. We report that the R-domains of NRPSs from Mycobacterium tuberculosis (RNRP) and Mycobacterium smegmatis (RGPL) do not strictly adhere to the obligatory mode of catalysis performed by SDRs, but instead can carry out reductive catalysis of substrate following random bi–bi reaction mechanism as deciphered by NMR and SAXS studies. The crucial conformational change associated with NADPH binding necessary to achieve catalytically competent conformation is also delineated by SAXS studies. Using ITC, we have demonstrated that mutation of catalytic tyrosine to phenylalanine in R-domains results in 3–4-fold decrease in affinity for NADPH and attribute this phenomenon to loss of the noncovalent cation–π interactions present between the tyrosine and nicotinamide ring. We propose that the adaptation to an alternative theme of bi–bi catalytic mechanism enables the R-domains to process the substrates transferred by upstream domains and maintain assembly-line enzymology.
    Journal of Structural Biology. 01/2014;
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The biological macromolecular world is homochiral and effective enforcement and perpetuation of this homochirality is essential for cell survival. In this study, we present the mechanistic basis of a configuration-specific enzyme that selectively removes D-amino acids erroneously coupled to tRNAs. The crystal structure of dimeric D-aminoacyl-tRNA deacylase (DTD) from Plasmodium falciparum in complex with a substrate-mimicking analog shows how it uses an invariant 'cross-subunit' Gly-cisPro dipeptide to capture the chiral centre of incoming D-aminoacyl-tRNA. While no protein residues are directly involved in catalysis, the unique side chain-independent mode of substrate recognition provides a clear explanation for DTD's ability to act on multiple D-amino acids. The strict chiral specificity elegantly explains how the enriched cellular pool of L-aminoacyl-tRNAs escapes this proofreading step. The study thus provides insights into a fundamental enantioselection process and elucidates a chiral enforcement mechanism with a crucial role in preventing D-amino acid infiltration during the evolution of translational apparatus. DOI: http://dx.doi.org/10.7554/eLife.01519.001.
    eLife Sciences 01/2013; 2:e01519.
  • [Show abstract] [Hide abstract]
    ABSTRACT: The proofreading function of aminoacyl-tRNA synthetases is crucial in maintaining the fidelity of protein synthesis. Most archaeal threonyl-tRNA synthetases (ThrRSs) possess a unique proofreading domain unrelated to their eukaryotic/bacterial counterpart. The crystal structure of this domain from the archaeon Pyrococcus abysii in complex with its cognate and noncognate substrate analogues had given insights into its catalytic and discriminatory mechanisms. To probe further into the mechanistic and evolutionary aspects of this domain, work has been extended to another archaeon Aeropyrum pernix. The organism possesses two proteins corresponding to threonyl-tRNA synthetase, i.e. ThrRS1 and ThrRS2, encoded by two different genes, thrS1 and thrS2, respectively. ThrRS1 is responsible for aminoacylation and ThrRS2 for proofreading activity. Here the purification, crystallization and preliminary X-ray crystallographic investigation of the N-terminal proofreading domain of ThrRS2 from A. pernix is reported. The crystals belong to either the P4(1)2(1)2 or P4(3)2(1)2 space group and consist of one monomer per asymmetric unit.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 11/2012; 68(Pt 11):1390-1393. · 0.55 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The folding and unfolding of structurally similar proteins belonging to a family have long been a focus of investigation of the structure-(un)folding relationship. Such studies are yet to reach a consensus about whether structurally similar domains follow common or different unfolding pathways. Members of the βγ-crystallin superfamily, which consists of structurally similar proteins with limited sequence similarity from diverse life forms spanning microbes to mammals, form an appropriate model system for exploring this relationship further. We selected a new member, Crybg3_D3, the third βγ-crystallin domain of non-lens vertebrate protein Crybg3 from mouse brain. The crystal structure determined at 1.86 Å demonstrates that the βγ-crystallin domain of Crybg3 resembles more closely the lens βγ-crystallins than the microbial crystallins do. However, interestingly, this structural cousin follows a quite distinct (un)folding pathway via formation of an intermediate state. The intermediate species is in a nativelike conformation with variation in flexibility and tends to form insoluble aggregates. The individual domains of lens βγ-crystallins (and microbial homologues) do not follow such an unfolding pattern. Thus, even the closest members of a subfamily within a superfamily do not necessarily follow similar unfolding paths, suggesting the divergence acquired by these domains, which could be observed only by unfolding. Additionally, this study provides insights into the modifications that this domain has undergone during its recruitment into the non-lens tissues in vertebrates.
    Biochemistry 10/2012; · 3.38 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The bacterial pathogen Xanthomonas oryzae pv. oryzae causes bacterial leaf blight, a serious disease of rice. The secreted exoglucanase CbsA is an important virulence factor of this pathogen. It belongs to the glycosyl hydrolase 6 family of proteins based on the carbohydrate-active enzyme (CAZY) classification. In this study, CbsA has been overexpressed, purified and crystallized. The crystal diffracted to a resolution of 1.86 Å and belonged to space group P2(1)2(1)2(1). It contained one monomer per asymmetric unit, with a solvent content of 45.8%.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2012; 68(Pt 10):1191-4. · 0.55 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In mycobacteria, polyketide synthases and nonribosomal peptide synthetases (NRPSs) produce complex lipidic metabolites by using a thio-template mechanism of catalysis. In this study, we demonstrate that off-loading reductase (R) domain of mycobacterial NRPSs performs two consecutive [2 + 2]e(-) reductions to release thioester-bound lipopeptides as corresponding alcohols, using a nonprocessive mechanism of catalysis. The first crystal structure of an R domain from Mycobacterium tuberculosis NRPS provides strong support to this mechanistic model and suggests that the displacement of intermediate would be required for cofactor recycling. We show that 4e(-) reductases produce alcohols through a committed aldehyde intermediate, and the reduction of this intermediate is at least 10 times more efficient than the thioester-substrate. Structural and biochemical studies also provide evidence for the conformational changes associated with the reductive cycle. Further, we show that the large substrate-binding pocket with a hydrophobic platform accounts for the remarkable substrate promiscuity of these domains. Our studies present an elegant example of the recruitment of a canonical short-chain dehydrogenase/reductase family member as an off-loading domain in the context of assembly-line enzymology.
    Proceedings of the National Academy of Sciences 03/2012; 109(15):5681-6. · 9.74 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Edited by J. E. Ladbury Keywords: βγ-crystallin; calcium binding; calcium-binding motif; N/D-N/D-X-X-S/T-S motif Numerous proteins belonging to the recently expanded βγ-crystallin superfamily bind Ca 2+ at the double-clamp N/D-N/D-X 1 -X 2 -S/T-S motif. However, there have been no attempts to understand the intricacies involving Ca 2+ binding, such as the determinants of Ca 2+ -binding affinity and their contributions to gain in stability. This work is an in-depth analysis of understanding the modes and determinants of Ca 2+ binding to βγ-crystallin motifs. We have performed extensive naturally occurring substitutions from related proteins on the βγ-crystallin domains of flavollin, a low-affinity Ca 2+ -binding protein, and clostrillin, a moderate-affinity protein. We monitored the consequences of these modifications on Ca 2 + binding by isothermal titration calorimetry, thermal stability and confor-mational and crystal structure analyses. We demonstrate that Ca 2 + binding to the two sites of a βγ-domain is interdependent and that the presence of Arg at the fifth position disables a site. A change from Thr to Ser, or vice versa, influences Ca 2+ -binding affinity, highlighting the basis of diversity found in these domains. A subtle change in the first site has a greater influence on Ca 2 + binding than a similar alteration in the second site. Thus, the second site is more variable in nature. Replacing an acidic or hydrophobic residue in a binding site alters the Ca 2+ -binding properties drastically. While it appears from their binding site sequence that these domains have evolved randomly, our examination illustrates the subtlety in the design of these modules. Decoding such design schemes would aid in our understanding of the functional themes underlying differential Ca 2 + binding and in predicting these in emerging sequence information.
    Journal of Molecular Biology 01/2012; · 3.91 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Activation of fatty acids as acyl-adenylates by fatty acyl-AMP ligases (FAALs) in Mycobacterium tuberculosis is a variant of a classical theme that involves formation of acyl-CoA (coenzyme A) by fatty acyl-CoA ligases (FACLs). Here, we show that FAALs and FACLs possess similar structural fold and substrate specificity determinants, and the key difference is the absence of a unique insertion sequence in FACL13 structure. A systematic analysis shows a conserved hydrophobic anchorage of the insertion motif across several FAALs. Strikingly, mutagenesis of two phenylalanine residues, which are part of the anchorage, to alanine converts FAAL32 to FACL32. This insertion-based in silico analysis suggests the presence of FAAL homologues in several other non-mycobacterial genomes including eukaryotes. The work presented here establishes an elegant mechanism wherein an insertion sequence drives the functional divergence of FAALs from canonical FACLs.
    Journal of Molecular Biology 12/2011; 416(2):221-38. · 3.91 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Leishmania META1 has for long been a candidate molecule for involvement in virulence: META1 transcript and protein are up-regulated in metacyclic Leishmania. Yet, how META1 contributes to virulence remains unclear. We sought insights into the possible functions of META1 by studying its evolutionary origins. Using multiple criteria including sequence similarity, nucleotide composition, phylogenetic analysis and selection pressure on gene sequence, we present evidence that META1 originated in trypanosomatids as a result of a lateral gene transfer of a bacterial heat-inducible protein, HslJ. Furthermore, within the Leishmania genome, META1 sequence is under negative selection pressure against change/substitution. Using homology modeling of Leishmania META1 based on solved NMR structure of HslJ, we show that META1 and HslJ share a similar structural fold. The best hit for other proteins with similar fold is MxiM, a protein involved in the type III secretion system in Shigella. The striking structural similarity shared by META1, HslJ and MxiM suggests a possibility of shared functions. Upon structural superposition with MxiM, we have observed a putative hydrophobic cavity in META1. Mutagenesis of select hydrophobic residues in this cavity affects the secretion of the secreted acid phosphatase (SAP), indicating META1's involvement in secretory processes in Leishmania. Overall, this work uses an evolutionary biology approach, 3D-modeling and site-directed mutagenesis to arrive at new insights into functions of Leishmania META1.
    BMC Evolutionary Biology 11/2011; 11:334. · 3.29 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Numerous proteins belonging to the recently expanded βγ-crystallin superfamily bind Ca(2+) at the double-clamp N/D-N/D-X(1)-X(2)-S/T-S motif. However, there have been no attempts to understand the intricacies involving Ca(2+) binding, such as the determinants of Ca(2+)-binding affinity and their contributions to gain in stability. This work is an in-depth analysis of understanding the modes and determinants of Ca(2+) binding to βγ-crystallin motifs. We have performed extensive naturally occurring substitutions from related proteins on the βγ-crystallin domains of flavollin, a low-affinity Ca(2+)-binding protein, and clostrillin, a moderate-affinity protein. We monitored the consequences of these modifications on Ca(2)(+) binding by isothermal titration calorimetry, thermal stability and conformational and crystal structure analyses. We demonstrate that Ca(2)(+) binding to the two sites of a βγ-domain is interdependent and that the presence of Arg at the fifth position disables a site. A change from Thr to Ser, or vice versa, influences Ca(2+)-binding affinity, highlighting the basis of diversity found in these domains. A subtle change in the first site has a greater influence on Ca(2)(+) binding than a similar alteration in the second site. Thus, the second site is more variable in nature. Replacing an acidic or hydrophobic residue in a binding site alters the Ca(2+)-binding properties drastically. While it appears from their binding site sequence that these domains have evolved randomly, our examination illustrates the subtlety in the design of these modules. Decoding such design schemes would aid in our understanding of the functional themes underlying differential Ca(2)(+) binding and in predicting these in emerging sequence information.
    Journal of Molecular Biology 10/2011; 415(1):75-91. · 3.91 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Rational and in vitro evolutionary approaches to improve either protein stability or aggregation resistance were successful, but empirical rules for simultaneous improvement of both stability and aggregation resistance under denaturing conditions are still to be ascertained. We have created a robust variant of a lipase from Bacillus subtilis named "6B" using multiple rounds of in vitro evolution. T(m) and optimum activity temperature of 6B is 78 °C and 65 °C, respectively, which is ~22 °C and 30 °C higher than that of wild-type lipase. Most significantly, 6B does not aggregate upon heating. Physical basis of remarkable thermostability and non-aggregating behavior of 6B was explored using X-ray crystallography, NMR and differential scanning calorimetry. Our structural investigations highlight the importance of tightening of mobile regions of the molecule such as loops and helix termini to attain higher thermostability. Accordingly, NMR studies suggest a very rigid structure of 6B lipase. Further investigation suggested that reduction/perturbation of the large hydrophobic patches present in the wild-type protein structure, decreased propensity of amino acid sequence for aggregation and absence of aggregation-prone intermediate during thermal unfolding of 6B can account for its resistance to aggregation. Overall, our study suggest that better anchoring of the loops with the rest of the protein molecule through mutations particularly on the sites that perturb/disturb the exposed hydrophobic patches can simultaneously increase protein stability and aggregation resistance.
    Journal of Molecular Biology 09/2011; 413(3):726-41. · 3.91 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: NGP-1(GNL-2) is a putative GTPase, overexpressed in breast carcinoma and localized in the nucleolus. NGP-1 belongs to the MMR1-HSR1 family of large GTPases that are emerging as crucial coordinators of signaling cascades in different cellular compartments. The members of this family share very closely related G-domains, but the signals and pathways regulating their subcellular localization and their functional relevance remain unknown. To improve our understanding of the nuclear transport mechanism of NGP-1, we have identified two nucleolar localization signals (NoLS) that are independently shown to translocate NGP-1 as well the heterologous protein to the nucleolus. Site-specific mutagenesis and immunofluorescence studies suggest that the tandem repeats of positively charged amino acids are critical for NGP-1 NoLS function. Interestingly, amino-terminal (NGP-1(1-100)) and carboxyl-terminal (NGP-1(661-731)) signals independently interact with receptors importin-β and importin-α, respectively. This investigation, for the first time, provides evidence that the interaction of importin-α with C-terminal NoLS (NGP-1(661-731)) was able to target the heterologous protein to the nucleolar compartment. Structural modeling analysis and alanine scanning mutagenesis of conserved G-domains suggest that G4 and G5 motifs are critical for GTP binding of NGP-1 and further show that the nucleolar localization of NGP-1 is regulated by a GTP gating-mediated mechanism. In addition, our data suggest that an ongoing transcription is essential for efficient localization of NGP-1 to the nucleolus. We have observed a high level of NGP-1 expression in the mitogen-activated primary human peripheral blood mononuclear cells (hPBMC) as well as in human fetal brain-derived neural precursor cells (hNPCs) in comparison to cells undergoing differentiation. Overall, the results suggest that multiple mechanisms are involved in the localization of NGP-1 to the nucleolus for the regulation of nucleolar function in cell growth and proliferation.
    Biochemistry 05/2011; 50(21):4521-36. · 3.38 Impact Factor
  • Debasisa Mohanty, Rajan Sankaranarayanan, Rajesh S Gokhale
    [Show abstract] [Hide abstract]
    ABSTRACT: The cell envelope of Mycobacterium tuberculosis (Mtb) possesses a repertoire of unusual lipids that are believed to play an important role in pathogenesis. In this review, we specifically focus on computational, biochemical and structural studies in lipid biosynthesis that have established functional role of polyketide synthases (PKSs) and fatty acyl-AMP ligases (FAALs). Mechanistic and structural studies with FAALs suggest that this group of proteins may have evolved from omnipresent fatty acyl-CoA ligases (FACLs). FAALs activate fatty acids as acyl-adenylates and transfer them on to the PKSs which then produce unusual acyl chains that are the components of mycobacterial lipids. FAALs are a newly discovered family of enzymes; whereas involvement of PKSs in lipid metabolism was not known prior to their discovery in Mtb. Since Mtb genome contains multiple homologs of FAALs and PKSs and owing to the conserved reaction mechanism and overlapping substrate specificity; there is tempting opportunity to develop 'systemic drugs' against these enzymes as anti-tuberculosis agents.
    Tuberculosis (Edinburgh, Scotland) 05/2011; 91(5):448-55. · 2.54 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Among the many different objectives of large scale structural genomics projects are expanding the protein fold space, enhancing understanding of a model or disease-related organism, and providing foundations for structure-based drug discovery. Systematic analysis of protein structures of Mycobacterium tuberculosis has been ongoing towards meeting some of these objectives. Indian participation in these efforts has been enthusiastic and substantial. The proteins of M. tuberculosis chosen for structural analysis by the Indian groups span almost all the functional categories. The structures determined by the Indian groups have led to significant improvement in the biochemical knowledge on these proteins and consequently have started providing useful insights into the biology of M. tuberculosis. Moreover, these structures form starting points for inhibitor design studies, early results of which are encouraging. The progress made by Indian structural biologists in determining structures of M. tuberculosis proteins is highlighted in this review.
    Tuberculosis (Edinburgh, Scotland) 04/2011; 91(5):456-68. · 2.54 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Editing/proofreading by aminoacyl-tRNA synthetases is an important quality control step in the accurate translation of the genetic code that removes noncognate amino acids attached to tRNA. Defects in the process of editing result in disease conditions including neurodegeneration. While proofreading, the cognate amino acids larger by a methyl group are generally thought to be sterically rejected by the editing modules as envisaged by the "Double-Sieve Model." Strikingly using solution based direct binding studies, NMR-heteronuclear single quantum coherence (HSQC) and isothermal titration calorimetry experiments, with an editing domain of threonyl-tRNA synthetase, we show that the cognate substrate can gain access and bind to the editing pocket. High-resolution crystal structural analyses reveal that functional positioning of substrates rather than steric exclusion is the key for the mechanism of discrimination. A strategically positioned "catalytic water" molecule is excluded to avoid hydrolysis of the cognate substrate using a "RNA mediated substrate-assisted catalysis mechanism" at the editing site. The mechanistic proof of the critical role of RNA in proofreading activity is a completely unique solution to the problem of cognate-noncognate selection mechanism.
    Proceedings of the National Academy of Sciences 12/2010; 107(51):22117-21. · 9.74 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Polycomb group (PcG) and trithorax group (trxG) proteins are chromatin-mediated regulators of a number of developmentally important genes including the homeotic genes. In Drosophila melanogaster, one of the trxG members, Trithorax like (Trl), encodes the essential multifunctional DNA binding protein called GAGA factor (GAF). While most of the PcG and trxG genes are conserved from flies to humans, a Trl-GAF homologue has been conspicuously missing in vertebrates. Here, we report the first identification of c-Krox/Th-POK as the vertebrate homologue of GAF on the basis of sequence similarity and comparative structural analysis. The in silico structural analysis of the zinc finger region showed preferential interaction of vertebrate GAF with GAGA sites similar to that of fly GAF. We also show by cross-immunoreactivity studies that both fly and vertebrate GAFs are highly conserved and share a high degree of structural similarity. Electrophoretic mobility shift assays show that vertebrate GAF binds to GAGA sites in vitro. Finally, in vivo studies by chromatin immunoprecipitation confirmed that vertebrate GAF binds to GAGA-rich DNA sequences present in hox clusters. Identification of vertebrate GAF and the presence of its target sites at various developmentally regulated loci, including hox complexes, highlight the evolutionarily conserved components involved in developmental mechanisms across the evolutionary lineage and answer a long-standing question of the presence of vertebrate GAF.
    Journal of Molecular Biology 07/2010; 400(3):434-47. · 3.91 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Proteins belonging to the lipocalin superfamily are usually secretory proteins of molecular mass approximately 20 kDa with a hydrophobic pocket for the binding and transport of diverse small ligands. Various lipocalins have been associated with many biological processes, e.g. immunomodulation, odorant transport, pheromonal activity, retinoid transport, cancer-cell interactions etc. However, the exact functions of many lipocalins and the ligands bound by them are unclear. Previously, the cDNA of a 20 kDa lipocalin (FLP) which is female-specifically expressed in the lacrimal glands of Syrian (golden) hamsters and secreted in the tears of females has been identified and cloned. His-tagged recombinant FLP (rFLP) has now been cloned, overexpressed in Escherichia coli as a soluble protein and purified to homogeneity using Ni-affinity followed by size-exclusion chromatography. Purified rFLP was crystallized using the sitting-drop vapour-diffusion method. The crystals tested belonged to space group P2(1)2(1)2(1) and diffracted to beyond 1.86 A resolution. Solvent-content analysis indicated the presence of one monomer in the asymmetric unit.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 05/2010; 66(Pt 5):509-12. · 0.55 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The βγ-crystallin superfamily consists of evolutionarily related proteins with domain topology similar to lens β-and γ-crystallins, formed from duplicated Greek key motifs. Ca 2þ binding was found in a few βγ-crystallin members earlier, although its prevalence and diversity as inherent molecular properties among members of the superfamily are not well studied. To increase our understanding of Ca 2þ binding in various βγ-crystallins, we undertook comprehensive structural and Ca 2þ -binding studies of seven members of the superfamily from bacteria, archaea, and vertebrates, including determination of high-resolution crystal structures of three proteins. Our structural observations show that the determinants of Ca 2þ coordination remain conserved in the form of an N/D-N/D-#-I-S/T-S motif in all domains. However, binding of Ca 2þ elicits varied physicochemical responses, ranging from passive sequestration to active stabilization. The motif in this superfamily is modified in some members like lens crystallins where Ca 2þ -binding abilities are partly or completely compromised. We show that reduction or loss of Ca 2þ binding in members of the superfamily, particularly in vertebrates, is due to the selective presence of unfavorable amino acids (largely Arg) at key Ca 2þ -ligation positions and that engineering of the canonical Ca 2þ -binding residues can confer binding activity on an otherwise inactive domain. Through this work, we demonstrate that βγ-crystallins with the N/ D-N/D-#-I-S/T-S motif form an extensive set of Ca 2þ -binding proteins prevalent in all of the three kingdoms of life. Ca 2þ binding is a crucial step in affecting major life pro-cesses (1) with specialized protein motifs engineered by nature to bind Ca 2þ with varied affinities in both the extracellular and intracellular environments (2). Proteins evolved to sense Ca 2þ as a signal are commonly referred to as sensors, and a set of proteins that bind and undergo structural stabilization are referred to as buffers. The all R-helical EF-hand superfamily of proteins and all β-sheet containing C2 domains predominate sensory Ca 2þ -bind-ing proteins. Proteins involved in structural binding are found in many extracellular proteins, such as EGF domains and cadher-ins, and are involved in providing structural stability to an organism by sequestering Ca 2þ from the environment. All of these independent structural units have been recruited as full-length proteins or as modules in multidomain proteins to perform Ca 2þ -dependent roles. Apart from these known families of Ca 2þ -binding proteins, some members of βγ-crystallins have been shown to bind Ca 2þ . The βγ-crystallin superfamily comprises proteins with β-sand-wich domains whose early characterized members, β-and γ-crystallins, are major components of the vertebrate eye lens (3-5). Individual βγ-crystallin domains are made of strand-exchanged Greek key motifs (6, 7). The wedge-like domains are characterized by the presence of highly ordered β-hairpin loops between the first and second strands of each motif. Two loops traverse the roof of the wedge-like domain and connect the two opposing β-pleated sheets of the domain (6-8). Evolutionarily related structural homologues of lens β-and γ-crystallins are found in some lower eukaryotic and prokaryotic species which together form the βγ-crystallin superfamily. The Ca 2þ -binding connection to the βγ-crystallin superfamily was observed more than 2 decades ago when protein S from Myxococcus xanthus, the first member of the superfamily, was shown to bind Ca 2þ even before the classification of the βγ-crystallin superfamily (7, 9, 10). At the same time, β-crystallin (aggregated form) from the vertebrate lens was shown to bind Ca 2þ albeit with low affinity in solution and with no structural evidence for the Ca 2þ -binding site (11). When the sequence of spherulin 3a from Physarum polycephalum was described with the putative features of βγ-crystallins as a third member, the concept of a superfamily, the βγ-crystallin superfamily originated (12). Spherulin 3a was structurally characterized and found to have bound Ca 2þ at the D/N-X-X-S motif, similar to that seen first in protein S (13-15). Later, a few members of this superfamily were demonstrated to bind Ca 2þ in solution (16-19). Protein S and spherulin 3a remained the only structurally well character-ized Ca 2þ -binding members of the βγ-crystallin superfamily
    Biochemistry 11/2009; · 3.38 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The betagamma-crystallin superfamily consists of evolutionarily related proteins with domain topology similar to lens beta- and gamma-crystallins, formed from duplicated Greek key motifs. Ca(2+) binding was found in a few betagamma-crystallin members earlier, although its prevalence and diversity as inherent molecular properties among members of the superfamily are not well studied. To increase our understanding of Ca(2+) binding in various betagamma-crystallins, we undertook comprehensive structural and Ca(2+)-binding studies of seven members of the superfamily from bacteria, archaea, and vertebrates, including determination of high-resolution crystal structures of three proteins. Our structural observations show that the determinants of Ca(2+) coordination remain conserved in the form of an N/D-N/D-#-I-S/T-S motif in all domains. However, binding of Ca(2+) elicits varied physicochemical responses, ranging from passive sequestration to active stabilization. The motif in this superfamily is modified in some members like lens crystallins where Ca(2+)-binding abilities are partly or completely compromised. We show that reduction or loss of Ca(2+) binding in members of the superfamily, particularly in vertebrates, is due to the selective presence of unfavorable amino acids (largely Arg) at key Ca(2+)-ligation positions and that engineering of the canonical Ca(2+)-binding residues can confer binding activity on an otherwise inactive domain. Through this work, we demonstrate that betagamma-crystallins with the N/D-N/D-#-I-S/T-S motif form an extensive set of Ca(2+)-binding proteins prevalent in all of the three kingdoms of life.
    Biochemistry 11/2009; 48(51):12180-90. · 3.38 Impact Factor
  • Source
    Gudlur Aparna, Avradip Chatterjee, Ramesh V Sonti, Rajan Sankaranarayanan
    [Show abstract] [Hide abstract]
    ABSTRACT: Xanthomonas oryzae pv oryzae (Xoo) causes bacterial blight, a serious disease of rice (Oryza sativa). LipA is a secretory virulence factor of Xoo, implicated in degradation of rice cell walls and the concomitant elicitation of innate immune responses, such as callose deposition and programmed cell death. Here, we present the high-resolution structural characterization of LipA that reveals an all-helical ligand binding module as a distinct functional attachment to the canonical hydrolase catalytic domain. We demonstrate that the enzyme binds to a glycoside ligand through a rigid pocket comprising distinct carbohydrate-specific and acyl chain recognition sites where the catalytic triad is situated 15 A from the anchored carbohydrate. Point mutations disrupting the carbohydrate anchor site or blocking the pocket, even at a considerable distance from the enzyme active site, can abrogate in planta LipA function, exemplified by loss of both virulence and the ability to elicit host defense responses. A high conservation of the module across genus Xanthomonas emphasizes the significance of this unique plant cell wall-degrading function for this important group of plant pathogenic bacteria. A comparison with the related structural families illustrates how a typical lipase is recruited to act on plant cell walls to promote virulence, thus providing a remarkable example of the emergence of novel functions around existing scaffolds for increased proficiency of pathogenesis during pathogen-plant coevolution.
    The Plant Cell 07/2009; 21(6):1860-73. · 9.25 Impact Factor

Publication Stats

987 Citations
330.22 Total Impact Points

Institutions

  • 2004–2014
    • Centre for Cellular and Molecular Biology
      Bhaganagar, Andhra Pradesh, India
  • 2009–2012
    • National Institute of Immunology
      New Dilli, NCT, India
    • University of Alberta
      • Department of Biochemistry
      Edmonton, Alberta, Canada
  • 2005–2009
    • IIT Kharagpur
      • Department of Biotechnology
      Kharagpur, Bengal, India
  • 2008
    • National Institutes of Health
      Maryland, United States
  • 2002–2008
    • Indian Institute of Science
      • Molecular Biophysics Unit
      Bengalore, State of Karnataka, India
  • 1999–2003
    • Institut de Génétique et de Biologie Moléculaire et Cellulaire
      Strasburg, Alsace, France
  • 2000–2001
    • French National Centre for Scientific Research
      Lutetia Parisorum, Île-de-France, France