Serological, genomic and structural analyses of the major mite allergen Der p 23.

BACKGROUND: Der p 23 was recently identified in a European population as a major allergen and potentially a chitin binding protein. OBJECTIVE: This study sought to assess the importance of Der p 23 among other Dermatophagoides allergens in a North American population and to determine the structure for functional characterization. METHODS: IgE binding to Der p 23, Der p 1, Der p 2, Der p 5, Der p 7 and Der p 8 was measured by ELISA. RNA-seq data from D. pteronyssinus were compared as estimates of allergen expression levels. The structure was analysed by X-ray crystallography and NMR. RESULTS: Despite a high prevalence of Der p 23, (75% vs. 87% and 79% for Der p 1 and Der p 2, respectively), the anti-Der p 23 IgE levels were relatively low. The patient response to the 6 allergens tested was variable (n = 47), but on average anti-Der p 1 and anti-Der p 2 together accounted for 85% of the specific IgE. In terms of abundance, the RNA expression level of Der p 23 is the lowest of the major allergens, thirty fold less than Der p 1 and sevenfold less than Der p 2. The structure of Der p 23 is a small, globular protein stabilized by two disulphide bonds, which is structurally related to allergens such as Blo t 12 that contain carbohydrate binding domains that bind chitin. Functional assays failed to confirm chitin binding by Der p 23. CONCLUSIONS AND CLINICAL RELEVANCE: Der p 23 accounts for a small percentage of the IgE response to mite allergens, which is dominated by Der p 1 and Der p 2. The prevalence and amount of specific IgE to Der p 23 and Der p 2 are disproportionately high compared to the expression of other Dermatophagoides allergens.

Ara h 2: crystal structure and IgE binding distinguish two subpopulations of peanut allergic patients by epitope diversity.

BACKGROUND: Peanut allergy affects 1% of the population and causes the most fatal food-related anaphylactic reactions. The protein Ara h 2 is the most potent peanut allergen recognized by 80-90% of peanut allergic patients. METHODS: The crystal structure of the major peanut allergen Ara h 2 was determined for the first time at 2.7 Å resolution using a customized maltose-binding protein (MBP)-fusion system. IgE antibody binding to the MBP fusion construct vs the natural allergen was compared by ELISA using sera from peanut allergic patients. RESULTS: The structure of Ara h 2 is a five-helix bundle held together by four disulfide bonds and related to the prolamin protein superfamily. The fold is most similar to other amylase and trypsin inhibitors. The MBP--Ara h 2 fusion construct was positively recognized by IgE from 76% of allergic patients (25/33). Two populations of patients could be identified. Subpopulation 1 (n = 14) showed an excellent correlation of IgE antibody binding to natural vs recombinant Ara h 2. Subpopulation 2 (n = 15) showed significantly reduced IgE binding to the MBP fusion protein. Interestingly, about 20% of the IgE binding in subpopulation 2 could be recovered by increasing the distance between MBP and Ara h 2 in a second construct. DISCUSSION: The reduced IgE binding to the MBP--Ara h 2 of subpopulation 2 indicates that the MBP molecule protects an immunodominant epitope region near the first helix of Ara h 2. Residues involved in the epitope(s) are suggested by the crystal structure. The MBP--Ara h 2 fusion constructs will be useful to further elucidate the relevance of certain epitopes to peanut allergy.

The corn inhibitor of blood coagulation factor XIIa. Crystallization and preliminary crystallographic analysis.

A 13.6 kDa protein from corn seeds is known to be a highly selective inhibitor of human blood coagulation Factor XIIa (or activated Hageman factor). We have crystallized this inhibitor at 23 degrees C and pH 7.5 from a solution of 30% polyethylene glycol 400, 0.2 M MgCl2, and 0.1 M Hepes. The crystals diffract to at least 2.1 A resolution. The space group is P4(2)2(1)2 with a = b = 57.15 A and c = 80.5 A. The crystals contain 51% solvent. Two heavy atom derivatives have been identified.

Transglutaminase factor XIII uses proteinase-like catalytic triad to crosslink macromolecules.

The X-ray crystal structure of human transglutaminase factor XIII has revealed a cysteine proteinase-like active site involved in a crosslinking reaction and not proteolysis. This is among the first observations of similar active sites in 2 different enzyme families catalyzing a similar reaction in opposite directions. Although the size and overall protein fold of factor XIII and the cysteine proteinases are quite different, the active site and the surrounding protein structure share structural features suggesting a common evolutionary lineage. Here we present a description of the residues in the active site and the structural evidence that the catalytic mechanism of the transglutaminases is similar to the reverse mechanism of the cysteine proteinases.

Crystal structure of SULT2A3, human hydroxysteroid sulfotransferase.

The crystal structure of SULT2A3 human hydroxysteroid sulfotransferase has been solved at 2.4 A resolution in the presence of 3'-phosphoadenosine 5'-phosphate (PAP). The overall structure is similar to those of SULT1 enzymes such as estrogen sulfotransferase and the PAP binding site is conserved, however, significant differences exist in the positions of loops Pro14-Ser20, Glu79-Ile82 and Tyr234-Gln244 in the substrate binding pocket. Moreover, protein interaction in the crystal structure has revealed a possible dimer-directed conformational alteration that may regulate the SULT activity.

The dimerization motif of cytosolic sulfotransferases.

Cytosolic sulfotransferases sulfate steroids such as estrogens and hydroxysteroids. The enzymes, including human estrogen sulfotransferase (hEST) and hydroxysteroid sulfotransferase (hHST), are generally homodimers in solution with mouse estrogen sulfotransferase (mEST) being one of few exceptions. To identify the amino acid residues responsible for the dimerization, eight residues on the surface of hEST were mutated to their counterparts in mEST and mutated hESTs were then analyzed by gel filtration chromatography. A single mutation of Val(269) to Glu was sufficient to convert hEST to a monomer and the corresponding mutation of Val(260) also altered hHST to a monomer. The hHST crystal structure revealed a short stretch of peptide with the side-chains from two hHST monomers forming a hydrophobic zipper-like structure enforced by ion pairs at both ends. This peptide consisted of 10 residues near the C-terminus that, including the critical Val residue, is conserved as KXXXTVXXXE in nearly all cytosolic sulfotransferases. When mEST underwent the double mutations Pro269Thr/Glu270Val dimerization resulted. Thus, the KXXXTVXXXE sequence appears to be the common protein-protein interaction motif that mediates the homo- as well as heterodimerization of cytosolic sulfotransferases.

A role of Lys614 in the sulfotransferase activity of human heparan sulfate N-deacetylase/N-sulfotransferase.

An active sulfotransferase (ST, residues 558-882) domain of the human heparan sulfate N-deacetylase/N-sulfotransferase (hHSNST) has been identified by aligning the amino acid sequence of hHSNST to that of mouse estrogen sulfotransferase (EST). The bacterially expressed ST domain transfers the 5'-sulfuryl group of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to only deacetylated heparin with an efficiency similar to that previously reported for the purified rat HSNST. Moreover, the K(m,PAPS) (2.1 microM) of the ST domain is also similar to that of the rat enzyme. Lys48 is a key residue in mEST catalysis. The residue corresponding to Lys48 is conserved in all known heparan sulfate sulfotransferases (Lys614 in the ST domain of hHSNST). Mutation of Lys614 to Ala abolishes N-sulfotransferase activity, indicating an important catalytic role of Lys614 in the ST domain. Crystals of the ST domain have been grown (orthorhombic space group P2(1)2(1)2) with diffraction to 2.5 A resolution.

Mouse steroid sulfotransferases: substrate specificity and preliminary X-ray crystallographic analysis.

Three mouse cytosolic sulfotransferases were expressed in Escherichia coli cells in order to study their substrate specificities toward natural as well as synthetic steroid hormones. The Km and Vmax values confirmed the high substrate specificity of estrogen and hydroxysteroid sulfotransferases toward estradiol and dehydroepiandrosterone, respectively. In sharp contrast, the synthetic estrogen diethylstilbestrol was metabolized efficiently by both enzymes to its disulfate ester. These sulfotransferases display highly stereospecific sulfotransferase activity for sulfating only the trans-isomer of diethylstilbestrol. Crystals suitable for high-resolution structure determination of estrogen sulfotransferase were grown with polyethylene glycol. The crystals belong to the orthorhombic space group P2(1)2(1)2, and diffracted to 2.5 A.

Structural evidence that the activation peptide is not released upon thrombin cleavage of factor XIII.

The three-dimensional structure of the recombinant human factor XIII a2 dimer after cleavage by thrombin has been determined by X-ray crystallography. Factor XIII zymogen was treated with bovine alpha-thrombin in the presence of 3 mM CaCl2, and the cleaved protein was crystallized from Tris buffered at pH 6.5 using ethanol as the precipitating agent. Refinement of the molecular model of thrombin-cleaved factor XIII against diffraction data from 10.0 to 2.5 A resolution has been carried out to give a crystallographic R factor of 18.2%. The structure of thrombin-cleaved factor XIII is remarkably similar to that of the zymogen: there are no large conformational changes in the protein and the 37 residue amino terminus activation peptide remains associated with the rest of the molecule. This work shows that the activation peptide, upon thrombin cleavage, has the same conformation and occupies the same position with respect to the rest of the molecule as it does in the zymogen structure.

Structural investigation of the antibiotic and ATP-binding sites in kanamycin nucleotidyltransferase.

Kanamycin nucleotidyltransferase (KNTase) is a plasmid-coded enzyme responsible for some types of bacterial resistance to aminoglycosides. The enzyme deactivates various antibiotics by transferring a nucleoside monophosphate group from ATP to the 4'-hydroxyl group of the drug. Detailed knowledge of the interactions between the protein and the substrates may lead to the design of aminoglycosides less susceptible to bacterial deactivation. Here we describe the structure of KNTase complexed with both the nonhydrolyzable nucleotide analog AMPCPP and kanamycin. Crystals employed in the investigation were grown from poly(ethylene glycol) solutions and belonged to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 57.3 A, b = 102.2 A, c = 101.8 A, and one dimer in the asymmetric unit. Least-squares refinement of the model at 2.5 A resolution reduced the crystallographic R factor to 16.8%. The binding pockets for both the nucleotide and the antibiotic are extensively exposed to the solvent and are composed of amino acid residues contributed by both subunits in the dimer. There are few specific interactions between the protein and the adenine ring of the nucleotide; rather the AMPCPP molecule is locked into position by extensive hydrogen bonding between the alpha-, beta-, and gamma-phosphates and protein side chains. This, in part, may explain the observation that the enzyme can utilize other nucleotides such as GTP and UTP. The 4'-hydroxyl group of the antibiotic is approximately 5 A from the alpha-phosphorus of the nucleotide and is in the proper orientation for a single in-line displacement attack at the phosphorus.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal structure of the sulfotransferase domain of human heparan sulfate N-deacetylase/ N-sulfotransferase 1.

Heparan sulfate N-deacetylase/N-sulfotransferase (HSNST) catalyzes the first and obligatory step in the biosynthesis of heparan sulfates and heparin. The crystal structure of the sulfotransferase domain (NST1) of human HSNST-1 has been determined at 2.3-A resolution in a binary complex with 3'-phosphoadenosine 5'-phosphate (PAP). NST1 is approximately spherical with an open cleft, and consists of a single alpha/beta fold with a central five-stranded parallel beta-sheet and a three-stranded anti-parallel beta-sheet bearing an interstrand disulfide bond. The structural regions alpha1, alpha6, beta1, beta7, 5'-phosphosulfate binding loop (between beta1 and alpha1), and a random coil (between beta8 and alpha13) constitute the PAP binding site of NST1. The alpha6 and random coil (between beta2 and alpha2), which form an open cleft near the 5'-phosphate of the PAP molecule, may provide interactions for substrate binding. The conserved residue Lys-614 is in position to form a hydrogen bond with the bridge oxygen of the 5'-phosphate.

Heparan/chondroitin sulfate biosynthesis. Structure and mechanism of human glucuronyltransferase I.

Human beta1,3-glucuronyltransferase I (GlcAT-I) is a central enzyme in the initial steps of proteoglycan synthesis. GlcAT-I transfers a glucuronic acid moiety from the uridine diphosphate-glucuronic acid (UDP-GlcUA) to the common linkage region trisaccharide Gal beta 1-3Gal beta 1-4Xyl covalently bound to a Ser residue at the glycosaminylglycan attachment site of proteoglycans. We have now determined the crystal structure of GlcAT-1 at 2.3 A in the presence of the donor substrate product UDP, the catalytic Mn(2+) ion, and the acceptor substrate analog Gal beta 1-3Gal beta 1-4Xyl. The enzyme is a alpha/beta protein with two subdomains that constitute the donor and acceptor substrate binding site. The active site residues lie in a cleft extending across both subdomains in which the trisaccharide molecule is oriented perpendicular to the UDP. Residues Glu(227), Asp(252), and Glu(281) dictate the binding orientation of the terminal Gal-2 moiety. Residue Glu(281) is in position to function as a catalytic base by deprotonating the incoming 3-hydroxyl group of the acceptor. The conserved DXD motif (Asp(194), Asp(195), Asp(196)) has direct interaction with the ribose of the UDP molecule as well as with the Mn(2+) ion. The key residues involved in substrate binding and catalysis are conserved in the glucuronyltransferase family as well as other glycosyltransferases.

Heparan sulphate N-sulphotransferase activity: reaction mechanism and substrate recognition.

Human heparan sulphate N-deacetylase/N-sulphotransferase 1 sulphates the NH(3) (+) group of the glucosamine moiety of the heparan chain in heparan sulphate/heparin biosynthesis. An open cleft that runs perpendicular to the sulphate donor 3'-phosphoadenosine 5'-phosphosulphate may constitute the acceptor substrate-binding site of the sulphotransferase domain (hNST1) [Kakuta, Sueyoshi, Negishi and Pedersen (1999) J. Biol. Chem. 274, 10673-10676]. When a hexasaccharide model chain is docked into the active site, only a trisaccharide (-IdoA-GlcN-IdoA-) portion interacts directly with the cleft residues: Trp-713, His-716 and His-720 from alpha helix 6, and Phe-640, Glu-641, Glu-642, Gln-644 and Asn-647 from random coil (residues 640-647). Mutation of these residues either abolishes or greatly reduces hNST1 activity. Glu-642 may play the critical role of catalytic base in the sulphuryl group transfer reaction, as indicated by its hydrogen-bonding distance to the NH(3) (+) group of the glucosamine moiety in the model and by mutational data.

Crystal structure of estrogen sulphotransferase.

The structure of estrogen sulphotransferase has been solved in the presence of inactive cofactor PAP and substrate 17 beta-estradiol. This structure reveals structural similarities between cytosolic sulphotransferases and nucleotide kinases.

Substrate gating confers steroid specificity to estrogen sulfotransferase.

Estrogen sulfotransferase (EST) exhibits a high substrate specificity and catalytic efficiency toward estrogens such as estradiol (E2) but insignificant ability to sulfate hydroxysteroids such as dehydroepiandrosterone (DHEA). To provide the structural basis for this estrogen specificity, we mutated amino acid residues that constitute the substrate-binding site of EST. Among these mutants, only Tyr-81 decreased E2 and increased DHEA sulfotransferase activities. Substitution for Tyr-81 by smaller hydrophobic residues increased K(m(E2)) for E2 activity, whereas the k(cat(E2)) remained relatively constant. The Y81L mutant exhibited the same DHEA activity as wild-type hydroxysteroid sulfotransferase, for which K(m(DHEA)) remained relatively constant, and k(cat(DHEA)) was markedly increased. The side chain of Tyr-81 is directed at the A-ring of the E2 molecule in the substrate-binding pocket of EST, constituting a steric gate with Phe-142 sandwiching E2 from the opposite side. The present mutagenesis study indicates that the 3beta-hydroxyl group of the DHEA molecule is excluded from the catalytic site of EST through steric hindrance of Tyr-81 with the C-19 methyl group of DHEA. Thus, this stricture-like gating caused by steric hindrance appears to be a structural principle for conferring estrogen specificity to EST.

The sulfuryl transfer mechanism. Crystal structure of a vanadate complex of estrogen sulfotransferase and mutational analysis.

Estrogen sulfotransferase (EST) catalyzes transfer of the 5'-sulfuryl group of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) to the 3alpha-phenol group of estrogenic steroids such as estradiol (E2). The recent crystal structure of EST-adenosine 3', 5'-diphosphate (PAP)- E2 complex has revealed that residues Lys48, Thr45, Thr51, Thr52, Lys106, His108, and Try240 are in position to play a catalytic role in the sulfuryl transfer reaction of EST (Kakuta Y., Pedersen, L. G., Carter, C. W., Negishi, M., and Pedersen, L. C. (1997) Nat. Struct. Biol. 4, 904-908). Mutation of Lys48, Lys106, or His108 nearly abolishes EST activity, indicating that they play a critical role in catalysis. A present 2.2-A resolution structure of EST-PAP-vanadate complex indicates that the vanadate molecule adopts a trigonal bipyramidal geometry with its equatorial oxygens coordinated to these three residues. The apical positions of the vanadate molecule are occupied by a terminal oxygen of the 5'-phosphate of PAP (2.1 A) and a possible water molecule (2. 3 A). This water molecule superimposes well to the 3alpha-phenol group of E2 in the crystal structure of the EST.PAP.E2 complex. These structures are characteristic of the transition state for an in-line sulfuryl transfer reaction from PAPS to E2. Moreover, residues Lys48, Lys106, and His108 are found to be coordinated with the vanadate molecule at the transition state of EST.

Crystal structure-based studies of cytosolic sulfotransferase.

Sulfation is a widely observed biological reaction conserved from bacterium to human that plays a key role in various biological processes such as growth, development, and defense against adversities. Deficiencies due to the lack of the ubiquitous sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) are lethal in humans. A large group of enzymes called sulfotransferases catalyze the transfer reaction of sulfuryl group of PAPS to the acceptor group of numerous biochemical and xenochemical substrates. Four X-ray crystal structures of sulfotransferases have now been determined: cytosolic estrogen, hydroxysteroid, aryl sulfotransferases, and a sulfotransferase domain of the Golgi-membrane heparan sulfate N-deacetylase/N-sulfotransferase 1. These have revealed the conserved core structure of the PAPS binding site, a common reaction mechanism, and some information concerning the substrate specificity. These crystal structures introduce a new era of the study of the sulfotransferases.

Heparan sulfate biosynthesis: a theoretical study of the initial sulfation step by N-deacetylase/N-sulfotransferase.

Heparan sulfate N-deacetylase/N-sulfotransferase (NDST) catalyzes the deacetylation and sulfation of N-acetyl-D-glucosamine residues of heparan sulfate, a key step in its biosynthesis. Recent crystallographic and mutational studies have identified several potentially catalytic residues of the sulfotransferase domain of this enzyme (, J. Biol. Chem. 274:10673-10676). We have used the x-ray crystal structure of heparan sulfate N-sulfotransferase with 3'-phosphoadenosine 5'-phosphate to build a solution model with cofactor 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and a model heparan sulfate ligand bound, and subsequently performed a 2-ns dynamics solution simulation. The simulation results confirm the importance of residues Glu(642), Lys(614), and Lys(833), with the possible involvement of Thr(617) and Thr(618), in binding PAPS. Additionally, Lys(676) is found in close proximity to the reaction site in our solvated structure. This study illustrates for the first time the possible involvement of water in the catalysis. Three water molecules were found in the binding site, where they are coordinated to PAPS, heparan sulfate, and the catalytic residues.