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Biomedical subjects

K R Acharya

Publications and source records attributed to K R Acharya.

At least 19 recordsLinked to original sources
PubMed2001

Structure of UDP complex of UDP-galactose:beta-galactoside-alpha -1,3-galactosyltransferase at 1.53-A resolution reveals a conformational change in the catalytically important C terminus.

UDP-galactose:beta-galactosyl alpha-1,3-galactosyltransferase (alpha3GT) catalyzes the transfer of galactose from UDP-alpha-d-galactose into an alpha-1,3 linkage with beta-galactosyl groups in glycoconjugates. The enzyme is expressed in many mammalian species but is absent from humans, apes, and old world monkeys as a result of the mutational inactivation of the gene; in humans, a large fraction of natural antibodies are directed against its product, the alpha-galactose epitope. alpha3GT is a member of a family of metal-dependent retaining glycosyltransferases including the histo-blood group A and B synthases. A crystal structure of the catalytic domain of alpha3GT was recently reported (Gastinel, L. N., Bignon, C., Misra, A. K., Hindsgaul, O., Shaper, J. H., and Joziasse, D. H. (2001) EMBO J. 20, 638-649). However, because of the limited resolution (2.3 A) and high mobility of the atoms (as indicated by high B-factors) this structure (form I) does not provide a clear depiction of the catalytic site of the enzyme. Here we report a new, highly ordered structure for the catalytic domain of alpha3GT at 1.53-A resolution (form II). This provides a more accurate picture of the details of the catalytic site that includes a bound UDP molecule and a Mn(2+) cofactor. Significantly, in the new structure, the C-terminal segment (residues 358-368) adopts a very different, highly structured conformation and appears to form part of the active site. The properties of an Arg-365 to Lys mutant indicate that this region is important for catalysis, possibly reflecting its role in a donor substrate-induced conformational change.

Amino Acid Sequence↗
PubMed2001

Cleavage of 3',5'-pyrophosphate-linked dinucleotides by ribonuclease A and angiogenin.

Recently, 3',5'-pyrophosphate-linked 2'-deoxyribodinucleotides were shown to be >100-fold more effective inhibitors of RNase A superfamily enzymes than were the corresponding monophosphate-linked (i.e., standard) dinucleotides. Here, we have investigated two ribo analogues of these compounds, cytidine 3'-pyrophosphate (P'-->5') adenosine (CppA) and uridine 3'-pyrophosphate (P'-->5') adenosine (UppA), as potential substrates for RNase A and angiogenin. CppA and UppA are cleaved efficiently by RNase A, yielding as products 5'-AMP and cytidine or uridine cyclic 2',3'-phosphate. The k(cat)/K(m) values are only 4-fold smaller than for the standard dinucleotides CpA and UpA, and the K(m) values (10-16 microM) are lower than those reported for any earlier small substrates (e.g., 500-700 microM for CpA and UpA). The k(cat)/K(m) value for CppA with angiogenin is also only severalfold smaller than for CpA, but the effect of lengthening the internucleotide linkage on K(m) is more modest. Ribonucleotide 3',5'-pyrophosphate linkages were proposed previously to exist in nature as chemically labile intermediates in the pathway for the generation of cyclic 2',3'-phosphate termini in various RNAs. We demonstrate that in fact they are relatively stable (t(1/2) > 15 days for uncatalyzed degradation of UppA at pH 6 and 25 degrees C) and that cleavage in vivo is most likely enzymatic. Replacements of the RNase A catalytic residues His12 and His119 by alanine reduce activity toward UppA by approximately 10(5)-and 10(3.3)-fold, respectively. Thus, both residues play important roles. His12 probably acts as a base catalyst in cleavage of UppA (as with RNA). However, the major function of His119 in RNA cleavage, protonation of the 5'-O leaving group, is not required for UppA cleavage because the pK(a) of the leaving group is much lower than that for RNA substrates. A crystal structure of the complex of RNase A with 2'-deoxyuridine 3'-pyrophosphate (P'-->5') adenosine (dUppA), determined at 1.7 A resolution, together with models of the UppA complex based on this structure suggest that His119 contributes to UppA cleavage through a hydrogen bond with a nonbridging oxygen atom in the pyrophosphate and through pi-pi stacking with the six-membered ring of adenine.

Alanine↗
PubMed2001

Crystallization and preliminary investigations on a telomeric repeat sequence C4A2C4A2.

Repeat units based on the telomeric sequence of the ciliated protozoan Tetrahymena, d(C(4)A(2))(2), have been crystallized. Cytosine-rich DNA stretches are known to reside in telomeres and centromeres of eukaryotic chromosomes, playing crucial roles in the structural stability of the chromosome in addition to their connection with cancer and aging. Preliminary investigations on the telomeric repeat sequence C(4)A(2)C(4)A(2) from CD studies and X-ray crystal data suggest it to be a right-handed interdigitated tetraplex structure with hemiprotonated C.C(+) base pairs. The molecules appear to be packed one on top of another forming a discontinuous helix along c simulating a poly-C fibre, an arrangement which maximizes the number of cytosines stacked.

Crystallization↗
PubMed2001

Crystal structure of the eosinophil major basic protein at 1.8 A. An atypical lectin with a paradigm shift in specificity.

The eosinophil major basic protein (EMBP) is the predominant constituent of the crystalline core of the eosinophil primary granule. EMBP is directly implicated in epithelial cell damage, exfoliation, and bronchospasm in allergic diseases such as asthma. Here we report the crystal structure of EMBP at 1.8 A resolution, and show that it is similar to that of members of the C-type lectin superfamily with which it shares minimal amino acid sequence identity (approximately 15--28%). However, this protein lacks a Ca(2+)/carbohydrate-binding site. Our analysis suggests that EMBP specifically binds heparin. Based on our results, we propose a possible new function for this protein, which is likely to have implications for EMBP function.

Blood Proteins↗
PubMed2001

Mapping the ribonucleolytic active site of eosinophil-derived neurotoxin (EDN). High resolution crystal structures of EDN complexes with adenylic nucleotide inhibitors.

Eosinophil-derived neurotoxin (EDN), a basic ribonuclease found in the large specific granules of eosinophils, belongs to the pancreatic RNase A family. Although its physiological function is still unclear, it has been shown that EDN is a neurotoxin capable of inducing the Gordon phenomenon in rabbits. EDN is also a potent helminthotoxin and can mediate antiviral activity of eosinophils against isolated virions of the respiratory syncytial virus. EDN is a catalytically efficient RNase sharing similar substrate specificity with pancreatic RNase A with its ribonucleolytic activity being absolutely essential for its neurotoxic, helminthotoxic, and antiviral activities. The crystal structure of recombinant human EDN in the unliganded form has been determined previously (Mosimann, S. C., Newton, D. L., Youle, R. J., and James, M. N. G. (1996) J. Mol. Biol. 260, 540-552). We have now determined high resolution (1.8 A) crystal structures for EDN in complex with adenosine-3',5'-diphosphate (3',5'-ADP), adenosine-2',5'-di-phosphate (2',5'-ADP), adenosine-5'-diphosphate (5'-ADP) as well as for a native structure in the presence of sulfate refined at 1.6 A. The inhibition constant of these mononucleotides for EDN has been determined. The structures present the first detailed picture of differences between EDN and RNase A in substrate recognition at the ribonucleolytic active site. They also provide a starting point for the design of tight-binding inhibitors, which may be used to restrain the RNase activity of EDN.

Adenosine Diphosphate↗
PubMed2001

High-level expression of three members of the murine angiogenin family in Escherichia coli and purification of the recombinant proteins.

Angiogenin (Ang) is a small basic protein which belongs to the pancreatic ribonuclease superfamily. It potently induces the formation of new blood vessels and has emerged as a promising anticancer target. Mice possess genes encoding one ortholog (mAng) and three homologs of Ang, designated angiogenin-related protein (mAngrp), angiogenin-3 (mAng-3), and angiogenin-4 (mAng-4). Structural and functional study of these homologs has been hampered by the low yield of protein from the existing heterologous expression system. In the experiments described, we used a pET expression vector to express these proteins in the cytoplasm of Escherichia coli BL21-CodonPlus(DE3)-RIL cells, whereupon substantial amounts of each accumulated in the form of insoluble aggregates. The proteins were renatured using an arginine-assisted procedure and subsequently purified by cation-exchange chromatography and reversed-phase HPLC; each purified protein was shown to be enzymatically active toward tRNA. The yields of pure mAngrp and mAng-3 were 7.6 and 12 mg/liter culture, respectively, representing substantial increases over previously reported experiments. This is also the first report of the expression and purification of mAng-4, obtained here in a yield of 30 mg/liter culture. The ready availability of milligram quantities of these proteins will enable further functional studies and high-resolution structural analyses to be conducted.

Angiogenesis Inducing Agents↗
PubMed2001

Crystal structure of Fab198, an efficient protector of the acetylcholine receptor against myasthenogenic antibodies.

The crystal structure of the Fab fragment of the rat monoclonal antibody 198, with protective activity for the main immunogenic region of the human muscle acetylcholine receptor against the destructive action of myasthenic antibodies, has been determined and refined to 2.8 A resolution by X-ray crystallographic methods. The mouse anti-lysozyme Fab D1.3 was used as a search model in molecular replacement with the AMORE software. The complementarity determining regions (CDR)-L2, CDR-H1 and CDR-H2 belong to canonical groups. Loops CDR-L3, CDR-H2 and CDR-H3, which seem to make a major contribution to binding, were analyzed and residues of potential importance for antigen-binding are examined. The antigen-binding site was found to be a long crescent-shaped crevice. The structure should serve as a model in the rational design of very high affinity humanized mutants of Fab198, appropriate for therapeutic approaches in the model autoimmune disease myasthenia gravis.

Amino Acid Sequence↗
PubMed2001

Binding of phosphate and pyrophosphate ions at the active site of human angiogenin as revealed by X-ray crystallography.

Human angiogenin (Ang) is an unusual homolog of bovine pancreatic RNase A that utilizes its ribonucleolytic activity to induce the formation of new blood vessels. The pyrimidine-binding site of Ang was shown previously to be blocked by glutamine 117, indicating that Ang must undergo a conformational change to bind and cleave RNA. The mechanism and nature of this change are not known, and no Ang-inhibitor complexes have been characterized structurally thus far. Here, we report crystal structures for the complexes of Ang with the inhibitors phosphate and pyrophosphate, and the structure of the complex of the superactive Ang variant Q117G with phosphate, all at 2.0 A resolution. Phosphate binds to the catalytic site of both Ang and Q117G in essentially the same manner observed in the RNase A-phosphate complex, forming hydrogen bonds with the side chains of His 13, His 114, and Gln 12, and the main chain of Leu 115; it makes an additional interaction with the Lys 40 ammonium group in the Ang complex. One of the phosphate groups of pyrophosphate occupies a similar position. The other phosphate extends toward Gln 117, and lies within hydrogen-bonding distance from the side-chain amide of this residue as well as the imidazole group of His 13 and the main-chain oxygen of Leu 115. The pyrimidine site remains obstructed in all three complex structures, that is, binding to the catalytic center is not sufficient to trigger the conformational change required for catalytic activity, even in the absence of the Gln 117 side chain. The Ang-pyrophosphate complex structure suggests how nucleoside pyrophosphate inhibitors might bind to Ang; this information may be useful for the design of Ang antagonists as potential anti-angiogenic drugs.

Binding Sites↗
PubMed2001

Role of conserved residues in structure and stability: tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily.

Serum retinol binding protein (RBP) is a member of the lipocalin family, proteins with up-and-down beta-barrel folds, low levels of sequence identity, and diverse functions. Although tryptophan 24 of RBP is highly conserved among lipocalins, it does not play a direct role in activity. To determine if Trp24 and other conserved residues have roles in stability and/or folding, we investigated the effects of conservative substitutions for the four tryptophans and some adjacent residues on the structure, stability, and spectroscopic properties of apo-RBP. Crystal structures of recombinant human apo-RBP and of a mutant with substitutions for tryptophans 67 and 91 at 1.7 A and 2.0 A resolution, respectively, as well as stability measurements, indicate that these relatively exposed tryptophans have little influence on structure or stability. Although Trp105 is largely buried in the wall of the beta-barrel, it can be replaced with minor effects on stability to thermal and chemical unfolding. In contrast, substitutions of three different amino acids for Trp24 or replacement of Arg139, a conserved residue that interacts with Trp24, lead to similar large losses in stability and lower yields of native protein generated by in vitro folding. The results and the coordinated nature of natural substitutions at these sites support the idea that conserved residues in functionally divergent homologs have roles in stabilizing the native relative to misfolded structures. They also establish conditions for studies of the kinetics of folding and unfolding by identifying spectroscopic signals for monitoring the formation of different substructures.

Carrier Proteins↗
PubMed2001

Structural features of a zinc binding site in the superantigen strepococcal pyrogenic exotoxin A (SpeA1): implications for MHC class II recognition.

Streptococcal pyrogenic exotoxin A (SpeA) is produced by Streptococcus pyogenes, and has been associated with severe infections such as scarlet fever and Streptococcal Toxic Shock Syndrome (STSS). In this study, the crystal structure of SpeA1 (the product of speA allele 1) in the presence of 2.5 mM zinc was determined at 2.8 A resolution. The protein crystallizes in the orthorhombic space group P2(1)2(1)2, with four molecules in the crystallographic asymmetric unit. The final structure has a crystallographic R-factor of 21.4% for 7,031 protein atoms, 143 water molecules, and 4 zinc atoms (one zinc atom per molecule). Four protein ligands-Glu 33, Asp 77, His 106, and His 110-form a zinc binding site that is similar to the one observed in a related superantigen, staphylococcoal enterotoxin C2. Mutant toxin forms substituting Ala for each of the zinc binding residues were generated. The affinity of these mutants for zinc ion confirms the composition of this metal binding site. The implications of zinc binding to SpeA1 for MHC class II recognition are explored using a molecular modeling approach. The results indicate that, despite their common overall architecture, superantigens appear to have multiple ways of complex formation with MHC class II molecules.

Alleles↗
PubMed2000

Crystal structures of apo- and holo-bovine alpha-lactalbumin at 2. 2-A resolution reveal an effect of calcium on inter-lobe interactions.

High affinity binding of Ca(2+) to alpha-lactalbumin (LA) stabilizes the native structure and is required for the efficient generation of native protein with correct disulfide bonds from the reduced denatured state. A progressive increase in affinity of LA conformers for Ca(2+) as they develop increasingly native structures can account for the tendency of the apo form to assume a molten globule state and the large acceleration of folding by Ca(2+). To investigate the effect of calcium on structure of bovine LA, x-ray structures have been determined for crystals of the apo and holo forms at 2.2-A resolution. In both crystal forms, which were grown at high ionic strength, the protein is in a similar global native conformation consisting of alpha-helical and beta-subdomains separated by a cleft. Even though alternative cations and Ca(2+) liganding solvent molecules are absent, removal of Ca(2+) has only minor effects on the structure of the metal-binding site and a structural change was observed in the cleft on the opposite face of the molecule adjoining Tyr(103) of the helical lobe and Gln(54) of the beta-lobe. Changes include increased separation of the lobes, loss of a buried solvent molecule near the Ca(2+)-binding site, and the replacement of inter- and intra-lobe H-bonds of Tyr(103) by interactions with new immobilized water molecules. The more open cleft structure in the apo protein appears to be an effect of calcium binding transmitted via a change in orientation of helix H3 relative to the beta-lobe to the inter-lobe interface. Calcium is well known to promote the folding of LA. The results from the comparison of apo and holo structures of LA provide high resolution structural evidence that the acceleration of folding by Ca(2+) is mediated by an effect on interactions between the two subdomains.

Amino Acid Sequence↗
PubMed2000

The crystal structure of human placenta growth factor-1 (PlGF-1), an angiogenic protein, at 2.0 A resolution.

The angiogenic molecule placenta growth factor (PlGF) is a member of the cysteine-knot family of growth factors. In this study, a mature isoform of the human PlGF protein, PlGF-1, was crystallized as a homodimer in the crystallographic asymmetric unit, and its crystal structure was elucidated at 2.0 A resolution. The overall structure of PlGF-1 is similar to that of vascular endothelial growth factor (VEGF) with which it shares 42% amino acid sequence identity. Based on structural and biochemical data, we have mapped several important residues on the PlGF-1 molecule that are involved in recognition of the fms-like tyrosine kinase receptor (Flt-1, also known as VEGFR-1). We propose a model for the association of PlGF-1 and Flt-1 domain 2 with precise shape complementarity, consider the relevance of this assembly for PlGF-1 signal transduction, and provide a structural basis for altered specificity of this molecule.

Amino Acid Sequence↗
PubMed2000

Microbial superantigens: from structure to function.

Superantigens are highly potent immune stimulators with a unique ability to interact simultaneously with MHC class II molecules and T cell receptors, forming a trimolecular complex that induces profound T-cell proliferation and massive cytokine production. Recent structural studies have provided a wealth of information regarding these complex interactions, and it is now emerging that, despite their common 3-D architecture, superantigens are able to crosslink MHC class II molecules and T cell receptors in a variety of ways.

Amino Acid Motifs↗
PubMed2000

The crystal structure of the Fab fragment of a rat monoclonal antibody against the main immunogenic region of the human muscle acetylcholine receptor.

The crystal structure of the Fab fragment of a rat monoclonal antibody, number 192, with a very high affinity (Kd = 0.05 nM) for the main immunogenic region of the human muscle acetylcholine receptor (AChR), has been determined and refined to 2.4 A resolution by X-ray crystallographic methods. The overall structure is similar to a Fab (NC6.8) from a murine antibody, used as a search model in molecular replacement. Structural comparisons with known antibody structures showed that the conformations of the hypervariable regions H1, H2, L1, L2, L3 of Fab192 adopt the canonical structures 1, 1, 2, 1, and 1, respectively. The surface of the antigen-binding site is relatively planar, as expected for an antibody against a large protein antigen, with an accessible area of 2865 A2. Analysis of the electrostatic surface potential of the antigen-binding site shows that the bottom of the cleft formed in the center of the site appears to be negatively charged. The structure will be useful in the rational design of very high affinity humanized mutants of Fab192, appropriate for therapeutic approaches of the model autoimmune disease myasthenia gravis.

Animals↗
PubMed2000

Structural similarities and differences in Staphylococcus aureus exfoliative toxins A and B as revealed by their crystal structures.

Staphylococcal aureus epidermolytic toxins (ETs) A and B are responsible for the induction of staphylococcal scalded skin syndrome, a disease of neonates and young children. The clinical features of this syndrome vary from localized blisters to severe exfoliation affecting most of the body surface. Comparison of the crystal structures of two subtypes of ETs-rETA (at 2.0 A resolution), rETB (at 2.8 A resolution), and an active site variant of rETA, Ser195Ala at 2.0 A resolution has demonstrated that their overall topology resembles that of a "trypsin-like" serine protease, but with significant differences at the N- and C-termini and loop regions. The details of the catalytic site in both ET structures are very similar to those in glutamate-specific serine proteases, suggesting a common catalytic mechanism. However, the "oxyanion hole," which is part of the catalytic sites of glutamate specific serine proteases, is in the closed or inactive conformation for rETA, yet in the open or active conformation for rETB. The ETs contain a unique amphipathic helix at the N-terminus, and it appears to be involved in optimizing the conformation of the catalytic site residues. Determination of the structure of the rETA catalytic site variant, Ser195Ala, showed no significant perturbation at the active site, establishing that the loss of biological and esterolytic activity can be attributed solely to disruption of the catalytic serine residue. Finally, the crystal structure of ETs, together with biochemical data and mutagenesis studies, strongly confirms the classification of these molecules as "serine proteases" rather than "superantigens."

Amino Acid Sequence↗
PubMed1999

Crystal structure of eosinophil cationic protein at 2.4 A resolution.

Eosinophil cationic protein (ECP) is located in the matrix of the eosinophil's large specific granule and has marked toxicity for a variety of helminth parasites, hemoflagellates, bacteria, single-stranded RNA virus, and mammalian cells and tissues. It belongs to the bovine pancreatic ribonuclease A (RNase A) family and exhibits ribonucleolytic activity which is about 100-fold lower than that of a related eosinophil ribonuclease, the eosinophil-derived neurotoxin (EDN). The crystal structure of human ECP, determined at 2.4 A, is similar to that of RNase A and EDN. It reveals that residues Gln-14, His-15, Lys-38, Thr-42, and His-128 at the active site are conserved as in all other RNase A homologues. Nevertheless, evidence for considerable divergence of ECP is also implicit in the structure. Amino acid residues Arg-7, Trp-10, Asn-39, His-64, and His-82 appear to play a key part in the substrate specificity and low catalytic activity of ECP. The structure also shows how the cationic residues are distributed on the surface of the ECP molecule that may have implications for an understanding of the cytotoxicity of this enzyme.

Amino Acid Sequence↗
PubMed1999

Selective recognition of mannose by the human eosinophil Charcot-Leyden crystal protein (galectin-10): a crystallographic study at 1.8 A resolution.

The role(s) of the eosinophil Charcot-Leyden crystal (CLC) protein in eosinophil or basophil function or associated inflammatory processes is yet to be established. Although the CLC protein has been reported to exhibit weak lysophospholipase activity, it shows virtually no sequence homology to any known member of this family of enzymes. The X-ray crystal structure of the CLC protein is very similar to the structure of the galectins, members of a beta-galactoside-specific animal lectin family, including a partially conserved galectin carbohydrate recognition domain (CRD). In the absence of any known natural carbohydrate ligand for this protein, the functional role of the CLC protein (galectin-10) has remained speculative. Here we describe structural studies on the carbohydrate binding properties of the CLC protein and report the first structure of a carbohydrate in complex with the protein. Interestingly, the CLC protein demonstrates no affinity for beta-galactosides and binds mannose in a manner very different from those of other related galectins that have been shown to bind lactosamine. The partial conservation of residues involved in carbohydrate binding led to significant changes in the topology and chemical nature of the CRD, and has implications for carbohydrate recognition by the CLC protein in vivo and its functional role in the biology of inflammation.

Acetylglucosamine↗