Structure and Carbohydrate Recognition by the Nonmitogenic Lectin Horcolin.

Lectins are sugar-binding proteins that have shown considerable promise as antiviral agents because of their ability to interact with envelope glycoproteins present on the surface of viruses such as HIV-1. However, their therapeutic potential has been compromised by their mitogenicity that stimulates uncontrolled division of T-lymphocytes. Horcolin, a member of the jacalin family of lectins, tightly binds the HIV-1 envelope glycoprotein gp120 and neutralizes HIV-1 particles but is nonmitogenic. In this report, we combine X-ray crystallography and NMR spectroscopy to obtain atomic-resolution insights into the structure of horcolin and the molecular basis for its carbohydrate recognition. Each protomer of the horcolin dimer adopts a canonical ß-prism I fold with three Greek key motifs and carries two carbohydrate-binding sites. The carbohydrate molecule binds in a negatively charged pocket and is stabilized by backbone and side chain hydrogen bonds to conserved residues in the ligand-binding loop. NMR titrations reveal a two-site binding mode and equilibrium dissociation constants for the two binding sites determined from two-dimensional (2D) lineshape modeling are 4-fold different. Single-binding-site variants of horcolin confirm the dichotomy in binding sites and suggest that there is allosteric communication between the two sites. An analysis of the horcolin structure shows a network of hydrogen bonds linking the two carbohydrate-binding sites directly and through a secondary binding site, and this coupling between the two sites is expected to assume importance in the interaction of horcolin with high-mannose glycans found on viral envelope glycoproteins.

Network of Entamoeba histolytica HSP18.5 dimers formed by two overlapping [IV]-X-[IV] motifs.

Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones with low molecular weight that prevent the aggregation of proteins during stress conditions and maintain protein homeostasis in the cell. sHSPs exist in dynamic equilibrium as a mixture of oligomers of various sizes with a constant exchange of subunits between them. Many sHSPs form cage-like assemblies that may dissociate into smaller oligomers during stress conditions. We carried out the functional and structural characterization of a small heat shock protein, HSP18.5, from Entamoeba histolytica (EhHSP18.5). It showed a pH-dependent change in its oligomeric state, which varied from a tetramer to larger than 48-mer. EhHSP18.5 protected Nde I and lysozyme substrates from temperature and chemical stresses, respectively. The crystal structure of EhHSP18.5 was determined at a resolution of 3.28 Å in C2221 cell with four subunits in the asymmetric unit forming two non-metazoan sHSP-type dimers. Unlike the reported cage-like structures, EhHSP18.5 formed a network of linear chains of molecules in the crystal. Instead of a single [IV]-X-[IV] motif, EhHSP18.5 has two overlapping I/V-X-I/V sequences at the C-terminus giving rise to novel interactions between the dimers. Negative staining Electron Microscopy images of EhHSP18.5 showed the presence of multiple oligomers: closed structures of various sizes and long tube-like structures.

Purification, characterization, and crystal structure of YhdA-type azoreductase from Bacillus velezensis.

Azoreductases are being extensively investigated for their ability to initiate degradation of recalcitrant azo dyes through reduction of azo bonds. There is great interest in studying their diversity, structure, and function to facilitate better understanding and effective application. Current study reports azoreductase enzyme from Bacillus velezensis, which showed 69.5% identity to the Bacillus subtilis azoreductase YhdA. The enzyme was homotetrameric and molecular weight of each subunit was 20 kDa. It decolorized azo dyes with different structures. The Vmax for decolorization of congo red, methyl orange and methyl red was 14.7, 28.6, and 77.9 nmol/min/mg, respectively. The enzyme contained FMN as cofactor and used NADPH as the favored co-substrate. It was oxygen-insensitive, but the presence of reducing agents enhanced its activity, which is a new finding. The azoreductase expression in B. velezensis was found to be unaffected by addition of azo dyes, although azo dyes are known to induce azoreductase expression in few organisms. The enzyme was thermostable with melting temperature of 89.5°C and functioned in wide temperature range. Further, the enzyme was crystallized and its structure was solved. The structural basis of its functional attributes is discussed. In our knowledge, this is the first report on characterization of azoreductase enzyme from B. velezensis.

Structural and related studies on Mevo lectin from Methanococcus voltae A3: the first thorough characterization of an archeal lectin and its interactions.

Crystallographic and solution studies of Mevo lectin and its complexes, the first effort of its kind on an archeal lectin, reveal a structure similar to ß-prism I fold lectins from plant and animal sources, but with a quaternary association involving a ring structure with seven-fold symmetry. Each subunit in the heptamer carries one sugar binding site on the first Greek key motif. The oligomeric interface is primarily made up of a parallel ß-sheet involving a strand of Greek key I of one subunit and Greek key ΙΙΙ from a neighboring subunit. The crystal structures of the complexes of the lectin with mannose, αMan(1,2)αMan, αMan(1,3)αMan, a mannotriose and a mannopentose revealed a primary binding site similar to that found in other mannose specific ß-prism I fold lectins. The complex with αMan(1,3)αMan provides an interesting case in which a few subunits have the reducing end at the primary binding site, while the majority have the nonreducing end at the primary binding site. The structures of complexes involving the trisaccharide and the pentasaccharide exhibit cross-linking among heptameric molecules. The observed arrangements may be relevant to the multivalency of the lectin. Phylogenetic analysis of amino acid sequences indicates that Mevo lectin is closer to ß-prism I fold animal lectins than with those of plant origin. The results presented here reinforce the conclusion regarding the existence of lectins in all three domains of life. It would also appear that lectins evolved to the present form before the three domains diverged.

Dodecameric structure of a small heat shock protein from Mycobacterium marinum M.

Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones present ubiquitously in all kingdoms of life. Their low molecular weight subunits associate to form higher order structures. Under conditions of stress, sHSPs prevent aggregation of substrate proteins by undergoing rapid changes in their conformation or stoichiometry. Polydispersity and dynamic nature of these proteins have made structural investigations through crystallography a daunting task. In pathogens like Mycobacteria, sHSPs are immuno-dominant antigens, enabling survival of the pathogen within the host and contributing to disease persistence. We characterized sHSPs from Mycobacterium marinum M and determined the crystal structure of one of these. The protein crystallized in three different conditions as dodecamers, with dimers arranged in a tetrahedral fashion to form a closed cage-like architecture. Interestingly, we found a pentapeptide bound to the dodecamers revealing one of the modes of sHSP-substrate interaction. Further, we have observed that ATP inhibits the chaperoning activity of the protein.

Structural and functional characterization of mercuric reductase from Lysinibacillus sphaericus strain G1.

In response to the widespread presence of inorganic Hg in the environment, bacteria have evolved resistance systems with mercuric reductase (MerA) as the key enzyme. MerA enzymes have still not been well characterized from gram positive bacteria. Current study reports physico-chemical, kinetic and structural characterization of MerA from a multiple heavy metal resistant strain of Lysinibacillus sphaericus, and discusses its implications in bioremediation application. The enzyme was homodimeric with subunit molecular weight of about 60 kDa. The Km and Vmax were found to be 32 µM of HgCl2 and 18 units/mg respectively. The enzyme activity was enhanced by ß-mercaptoethanol and NaCl up to concentrations of 500 µM and 100 mM respectively, followed by inhibition at higher concentrations. The enzyme showed maximum activity in the pH range of 7-7.5 and temperature range of 25-50 °C, with melting temperature of 67 °C. Cu2+ exhibited pronounced inhibition of the enzyme with mixed inhibition pattern. The enzyme contained FAD as the prosthetic group and used NADPH as the preferred electron donor, but it showed slight activity with NADH as well. Structural characterization was carried out by circular dichroism spectrophotometry and X-ray crystallography. X-ray confirmed the homodimeric structure of enzyme and gave an insight on the residues involved in catalytic binding. In conclusion, the investigated enzyme showed higher catalytic efficiency, temperature stability and salt tolerance as compared to MerA enzymes from other mesophiles. Therefore, it is proposed to be a promising candidate for Hg2+ bioremediation.

Autoinhibitory mechanism and activity-related structural changes in a mycobacterial adenylyl cyclase.

An adenylyl cyclase from Mycobacterium avium, Ma1120, is a functional orthologue of a pseudogene Rv1120c from Mycobacterium tuberculosis. We report the crystal structure of Ma1120 in a monomeric form and its truncated construct as a dimer. Ma1120 exists as a monomer in solution and crystallized as a monomer in the absence of substrate or inhibitor. An additional α-helix present at the N-terminus of the monomeric structure blocks the active site by interacting with the substrate binding residues and occupying the dimer interface region. However, the enzyme has been found to be active in solution, indicating the movement of the helix away from the interface to facilitate the formation of active dimers in conditions favourable for catalysis. Thus, the N-terminal helix of Ma1120 keeps the enzyme in an autoinhibited state when it is not active. Deletion of this helix enabled us to crystallize the molecule as an active homodimer in the presence of a P-site inhibitor 2',5'-dideoxy-3'-ATP, or pyrophosphate along with metal ions. The substrate specifying lysine residue plays a dual role of interacting with the substrate and stabilizing the dimer. The dimerization loop region harbouring the second substrate specifying residue, an aspartate, shows significant differences in conformation and position between the monomeric and dimeric structures. Thus, this study has not only revealed that significant structural transitions are required for the interconversion of the inactive and the active forms of the enzyme, but also provided precise nature of these transitions.

Affinity of a galactose-specific legume lectin from Dolichos lablab to adenine revealed by X-ray cystallography.

Crystal structure analysis of a galactose-specific lectin from a leguminous food crop Dolichos lablab (Indian lablab beans) has been carried out to obtain insights into its quaternary association and lectin-carbohydrate interactions. The analysis led to the identification of adenine binding sites at the dimeric interfaces of the heterotetrameric lectin. Structural details of similar adenine binding were reported in only one legume lectin, Dolichos biflorus, before this study. Here, we present the structure of the galactose-binding D. lablab lectin at different pH values in the native form and in complex with galactose and adenine. This first structure report on this lectin also provides a high resolution atomic view of legume lectin-adenine interactions. The tetramer has two canonical and two DB58-like interfaces. The binding of adenine, a non-carbohydrate ligand, is found to occur at four hydrophobic sites at the core of the tetramer at the DB58-like dimeric interfaces and does not interfere with the carbohydrate-binding site. To support the crystallographic observations, the adenine binding was further quantified by carrying out isothermal calorimetric titration. By this method, we not only estimated the affinity of the lectin to adenine but also showed that adenine binds with negative cooperativity in solution.

Structure and interactions of the phloem lectin (phloem protein 2) Cus17 from Cucumis sativus.

Phloem protein 2 (PP2) contributes crucially to phloem-based defense in plants by binding to carbohydrates displayed by pathogens. However, its three-dimensional structure and the sugar binding site remained unexplored. Here, we report the crystal structure of the dimeric PP2 Cus17 from Cucumis sativus in its apo form and complexed with nitrobenzene, N-acetyllactosamine, and chitotriose. Each protomer of Cus17 consists of two antiparallel four-stranded twisted ß sheets, a ß hairpin, and three short helices forming a ß sandwich architectural fold. This structural fold has not been previously observed in other plant lectin families. Structure analysis of the lectin-carbohydrate complexes reveals an extended carbohydrate binding site in Cus17, composed mostly of aromatic amino acids. Our studies suggest a highly conserved tertiary structure and a versatile binding site capable of recognizing motifs common to diverse glycans on plant pathogens/pests, which makes the PP2 family suited for phloem-based plant defense.

A new pentameric structure of rotavirus NSP4 revealed by molecular replacement.

The region spanning residues 95-146 of the rotavirus nonstructural protein NSP4 from the asymptomatic human strain ST3 has been purified and crystallized and diffraction data have been collected to a resolution of 2.6 Å. Several attempts to solve the structure by the molecular-replacement method using the available tetrameric structures of this domain were unsuccessful despite a sequence identity of 73% to the already known structures. A more systematic approach with a dimer as the search model led to an unexpected pentameric structure using the program Phaser. The various steps involved in arriving at this molecular-replacement solution, which unravelled a case of subtle variation between different oligomeric states unknown at the time of solving the structure, are presented in this paper.

Severe diffraction anisotropy, rotational pseudosymmetry and twinning complicate the refinement of a pentameric coiled-coil structure of NSP4 of rotavirus.

The crystal structure of the region spanning residues 95-146 of the rotavirus nonstructural protein NSP4 from the asymptomatic human strain ST3 was determined at a resolution of 2.5 Å. Severe diffraction anisotropy, rotational pseudosymmetry and twinning complicated the refinement of this structure. A systematic explanation confirming the crystal pathologies and describing how the structure was successfully refined is given in this report.

Novel pentameric structure of the diarrhea-inducing region of the rotavirus enterotoxigenic protein NSP4.

A novel pentameric structure which differs from the previously reported tetrameric form of the diarrhea-inducing region of the rotavirus enterotoxin NSP4 is reported here. A significant feature of this pentameric form is the absence of the calcium ion located in the core region of the tetrameric structures. The lysis of cells, the crystallization of the region spanning residues 95 to 146 of NSP4 (NSP4(95-146)) of strain ST3 (ST3:NSP4(95-146)) at acidic pH, and comparative studies of the recombinant purified peptide under different conditions by size-exclusion chromatography (SEC) and of the crystal structures suggested pH-, Ca(2+)-, and protein concentration-dependent oligomeric transitions in the peptide. Since the NSP4(95-146) mutant lacks the N-terminal amphipathic domain (AD) and most of the C-terminal flexible region (FR), to demonstrate that the pentameric transition is not a consequence of the lack of the N- and C-terminal regions, glutaraldehyde cross-linking of the ΔN72 and ΔN94 mutant proteins, which contain or lack the AD, respectively, but possess the complete C-terminal FR, was carried out. The results indicate the presence of pentamers in preparations of these longer mutants. Detailed SEC analyses of ΔN94 prepared under different conditions, however, revealed protein concentration-dependent but metal ion- and pH-independent pentamer accumulation at high concentrations which dissociated into tetramers and lower oligomers at low protein concentrations. While calcium appeared to stabilize the tetramer, magnesium in particular stabilized the dimer. ΔN72 existed primarily in the multimeric form under all conditions. These findings of a calcium-free NSP4 pentamer and its concentration-dependent and largely calcium-independent oligomeric transitions open up a new dimension in an understanding of the structural basis of its multitude of functions.

Structural and functional insights into the DNA damage-inducible protein 1 (Ddi1) from protozoa.

Ddi1 is a multidomain protein that belongs to the ubiquitin receptor family of proteins. The Ddi1 proteins contain a highly conserved retroviral protease (RVP)-like domain along with other domains. The severity of opportunistic infections, caused by parasitic protozoa in AIDS patients, was found to decline when HIV protease inhibitors were used in antiretroviral therapy. Parasite growth was shown to be suppressed by a few of the inhibitors targeting Ddi1 present in these parasites. In this study, the binding of HIV protease inhibitors to the RVP domain of Ddi1 from Toxoplasma gondii and Cryptosporidium hominis; and the binding of ubiquitin to the ubiquitin-associated domain of Ddi1 from these two parasites were established using Biolayer Interferometry. The crystal structures of the RVP domains of Ddi1 from T. gondii and C. hominis were determined; they form homodimers similar to those observed in HIV protease and the reported structures of the same domain from Saccharomyces cerevisiae, Leishmania major and humans. The native form of the domain showed an open dimeric structure and a normal mode analysis revealed that it can take up a closed conformation resulting from relative movements of the subunits. Based on the crystal structure of the RVP domain of Ddi1 from L. major, a seven residue peptide inhibitor was designed and it was shown to bind to the RVP domain of Ddi1 from L. major by Biolayer Interferometry. This peptide was modified using computational methods and was shown to have a better affinity than the initial peptide.

Structural basis for the functional and inhibitory mechanisms of ß-hydroxyacyl-acyl carrier protein dehydratase (FabZ) of Plasmodium falciparum.

The ß-hydroxyacyl-acyl carrier protein dehydratase of Plasmodium falciparum (PfFabZ) catalyzes the third and important reaction of the fatty acid elongation cycle. The crystal structure of PfFabZ is available in hexameric (active) and dimeric (inactive) forms. However, PfFabZ has not been crystallized with any bound inhibitors until now. We have designed a new condition to crystallize PfFabZ with its inhibitors bound in the active site, and determined the crystal structures of four of these complexes. This is the first report on any FabZ enzyme with active site inhibitors that interact directly with the catalytic residues. Inhibitor binding not only stabilized the substrate binding loop but also revealed that the substrate binding tunnel has an overall shape of "U". In the crystal structures, residue Phe169 located in the middle of the tunnel was found to be in two different conformations, open and closed. Thus, Phe169, merely by changing its side chain conformation, appears to be controlling the length of the tunnel to make it suitable for accommodating longer substrates. The volume of the substrate binding tunnel is determined by the sequence as well as by the conformation of the substrate binding loop region and varies between organisms for accommodating fatty acids of different chain lengths. This report on the crystal structures of the complexes of PfFabZ provides the structural basis of the inhibitory mechanism of the enzyme that could be used to improve the potency of inhibitors against an important component of fatty acid synthesis common to many infectious organisms.

Modification of the sugar specificity of a plant lectin: structural studies on a point mutant of Erythrina corallodendron lectin.

A mutant of Erythrina corallodendron lectin was generated with the aim of enhancing its affinity for N-acetylgalactosamine. A tyrosine residue close to the binding site of the lectin was mutated to a glycine in order to facilitate stronger interactions between the acetamido group of the sugar and the lectin which were prevented by the side chain of the tyrosine in the wild-type lectin. The crystal structures of this Y106G mutant lectin in complex with galactose and N-acetylgalactosamine have been determined. A structural rationale has been provided for the differences in the relative binding affinities of the wild-type and mutant lectins towards the two sugars based on the structures. A hydrogen bond between the O6 atom of the sugars and the variable loop of the carbohydrate-binding site of the lectin is lost in the mutant complexes owing to a conformational change in the loop. This loss is compensated by an additional hydrogen bond that is formed between the acetamido group of the sugar and the mutant lectin in the complex with N-acetylgalactosamine, resulting in a higher affinity of the mutant lectin for N-acetylgalactosamine compared with that for galactose, in contrast to the almost equal affinity of the wild-type lectin for the two sugars. The structure of a complex of the mutant with a citrate ion bound at the carbohydrate-binding site that was obtained while attempting to crystallize the complexes with sugars is also presented.

Effect of substrate binding loop mutations on the structure, kinetics, and inhibition of enoyl acyl carrier protein reductase from Plasmodium falciparum.

Enoyl acyl carrier protein reductase (ENR), which catalyzes the final and rate limiting step of fatty acid elongation, has been validated as a potential drug target. Triclosan is known to be an effective inhibitor for this enzyme. We mutated the substrate binding site residue Ala372 of the ENR of Plasmodium falciparum (PfENR) to Methionine and Valine which increased the affinity of the enzyme towards triclosan to almost double, close to that of Escherichia coli ENR (EcENR) which has a Methionine at the structurally similar position of Ala372 of PfENR. Kinetic studies of the mutants of PfENR and the crystal structure analysis of the A372M mutant revealed that a more hydrophobic environment enhances the affinity of the enzyme for the inhibitor. A triclosan derivative showed a threefold increase in the affinity towards the mutants compared to the wild type, due to additional interactions with the A372M mutant as revealed by the crystal structure. The enzyme has a conserved salt bridge which stabilizes the substrate binding loop and appears to be important for the active conformation of the enzyme. We generated a second set of mutants to check this hypothesis. These mutants showed loss of function, except in one case, where the crystal structure showed that the substrate binding loop is stabilized by a water bridge network.

Multiple nanocages of a cyanophage small heat shock protein with icosahedral and octahedral symmetries.

The structures of a cyanophage small heat shock protein (sHSP) were determined as octahedrons of 24-mers and 48-mers and as icosahedrons of 60-mers. An N-terminal deletion construct of an 18 kDa sHSP of Synechococcus sp. phage S-ShM2 crystallized as a 24-mer and its structure was determined at a resolution of 7 Å. The negative stain electron microscopy (EM) images showed that the full-length protein is a mixture of a major population of larger and a minor population of smaller cage-like particles. Their structures have been determined by electron cryomicroscopy 3D image reconstruction at a resolution of 8 Å. The larger particles are 60-mers with icosahedral symmetry and the smaller ones are 48-mers with octahedral symmetry. These structures are the first of the viral/phage origin and the 60-mer is the largest and the first icosahedral assembly to be reported for sHSPs.

X-ray crystallographic analysis of the complexes of enoyl acyl carrier protein reductase of Plasmodium falciparum with triclosan variants to elucidate the importance of different functional groups in enzyme inhibition.

Triclosan, a well-known inhibitor of Enoyl Acyl Carrier Protein Reductase (ENR) from several pathogenic organisms, is a promising lead compound to design effective drugs. We have solved the X-ray crystal structures of Plasmodium falciparum ENR in complex with triclosan variants having different substituted and unsubstituted groups at different key functional locations. The structures revealed that 4 and 2' substituted compounds have more interactions with the protein, cofactor, and solvents when compared with triclosan. New water molecules were found to interact with some of these inhibitors. Substitution at the 2' position of triclosan caused the relocation of a conserved water molecule, leading to an additional hydrogen bond with the inhibitor. This observation can help in conserved water-based inhibitor design. 2' and 4' unsubstituted compounds showed a movement away from the hydrophobic pocket to compensate for the interactions made by the halogen groups of triclosan. This compound also makes additional interactions with the protein and cofactor which compensate for the lost interactions due to the unsubstitution at 2' and 4'. In cell culture, this inhibitor shows less potency, which indicates that the chlorines at 2' and 4' positions increase the ability of the inhibitor to cross multilayered membranes. This knowledge helps us to modify the different functional groups of triclosan to get more potent inhibitors.

Analysis of proteins with the 'hot dog' fold: prediction of function and identification of catalytic residues of hypothetical proteins.

BACKGROUND: The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. RESULTS: We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. CONCLUSION: The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.

Crystal structure of the legume lectin-like domain of an ERGIC-53-like protein from Entamoeba histolytica.

ERGIC-53-like proteins are type I membrane proteins that belong to the class of intracellular cargo receptors and are known to be indispensable for the intracellular transport of glycoproteins. They are implicated in transporting glycoproteins between the endoplasmic reticulum and the Golgi body. The crystal structure of the legume lectin-like domain of an ERGIC-53-like protein from Entamoeba histolytica has been determined at 2.4 Šresolution. Although the overall structure of the domain resembles those of its mammalian and yeast orthologs (ERGIC-53 and Emp46, respectively), there are significant changes in the carbohydrate-binding site. A sequence-based search revealed the presence of several homologs of ERGIC-53 in different species of Entamoeba. This is the first report of the structural characterization of a member of this class of proteins from a protozoan and serves to further knowledge and understanding regarding the species-specific differences.