The structure of the mite allergen Blo t 1 explains the limited antibody cross-reactivity to Der p 1.

The Blomia tropicalis (Blo t) mite species is considered a storage mite in temperate climate zones and an important source of indoor allergens causing allergic asthma and rhinitis in tropical and subtropical regions. Here, we report the crystal structure of one of the allergens from Blo t, recombinant proBlo t 1 (rproBlo t 1), determined at 2.1 Å resolution. Overall, the fold of rproBlo t 1 is characteristic for the pro-form of cysteine proteases from the C1A class. Structural comparison of experimentally mapped Der f 1/Der p1 IgG epitopes to the same surface patch on Blo t 1, as well as of sequence identity of surface-exposed residues, suggests limited cross-reactivity between these allergens and Blo t 1. This is in agreement with ELISA inhibition results showing that, although cross-reactive human IgE epitopes exist, there are unique IgE epitopes for both Blo t 1 and Der p 1.

A novel dualistic profile of an allosteric AMPA receptor modulator identified through studies on recombinant receptors, mouse hippocampal synapses and crystal structures.

Positive allosteric modulators (PAMs) of 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors receive increasing interest as therapeutic drugs and have long served as important experimental tools in the study of the molecular mechanisms underlying glutamate-mediated neurotransmission. The aim of this study was to investigate functional and structural aspects of a novel analog of the AMPA receptor PAM cyclothiazide (CTZ) on recombinant and native glutamate receptors. We expressed rat GluA4flip and flop in Xenopus oocytes and characterized NS1376 and CTZ under two-electrode voltage-clamp. The dose-response analyses revealed dual effects of NS1376. The modulator induced 30-fold and 42-fold reductions in glutamate potency and increased the glutamate efficacy by 3.2-fold and 5.3-fold at GluA4flip and GluA4flop, respectively. Rapid application of glutamate to excised outside-out patches showed that NS1376 markedly attenuated desensitization, supporting the increased efficacy observed in the oocytes. Furthermore, when applied to acutely isolated mouse brain slices, NS1376 reduced the field excitatory postsynaptic potentials (fEPSPs) in the hippocampus to 51.6 ± 4.3% of baseline, likely as a consequence of reduced glutamate potency. However, the modulator displayed no effects on a sub-maximal long-term potentiation (LTP) protocol. We confirmed that CTZ increases presynaptic transmitter release, a property which was not shared by NS1376. Finally, we obtained detailed molecular information through X-ray structures, docking and molecular dynamics, which revealed that NS1376 interacts at the dimer interface of the ligand-binding domain in a manner overall similar to CTZ. NS1376 reveals that minor structural changes in CTZ can result in an altered modulatory profile, both enhancing agonist efficacy while markedly reducing agonist potency. These unique properties add new aspects to the complexity of allosteric modulations in neuronal systems.

The influence of a chiral amino acid on the helical handedness of PNA in solution and in crystals.

The X-ray structure of a self-complementary PNA hexamer (H-CGTACG-L-Lys-NH(2)) has been determined to 2.35 A resolution. The introduction of an L-lysine moiety has previously been shown to induce a preferred left-handedness of the PNA double helices in aqueous solution. However, in the crystal structure an equal amount of interchanging right- and left-handed helices is observed. The lysine moieties are pointing into large solvent channels and no significant interactions between this moiety and the remaining PNA molecule are observed. In contrast, molecular mechanics calculations show a preference for the left-handed helix of this hexameric PNA in aqueous solution as expected. The calculations indicate that the difference in the free energy of solvation between the left-handed and the right-handed helix is the determining factor for the preference of the left-handed helix in aqueous solution.

GluR2 ligand-binding core complexes: importance of the isoxazolol moiety and 5-substituent for the binding mode of AMPA-type agonists.

X-ray structures of the GluR2 ligand-binding core in complex with (S)-Des-Me-AMPA and in the presence and absence of zinc ions have been determined. (S)-Des-Me-AMPA, which is devoid of a substituent in the 5-position of the isoxazolol ring, only has limited interactions with the partly hydrophobic pocket of the ligand-binding site, and adopts an AMPA-like binding mode. The structures, in comparison with other agonist complex structures, disclose the relative importance of the isoxazolol ring and of the substituent in the 5-position for the mode of binding. A relationship appears to exist between the extent of interaction of the ligand with the hydrophobic pocket and the affinity of the ligand.

Structural basis for AMPA receptor activation and ligand selectivity: crystal structures of five agonist complexes with the GluR2 ligand-binding core.

Glutamate is the principal excitatory neurotransmitter within the mammalian CNS, playing an important role in many different functions in the brain such as learning and memory. In this study, a combination of molecular biology, X-ray structure determinations, as well as electrophysiology and binding experiments, has been used to increase our knowledge concerning the ionotropic glutamate receptor GluR2 at the molecular level. Five high-resolution X-ray structures of the ligand-binding domain of GluR2 (S1S2J) complexed with the three agonists (S)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol-4-yl]propionic acid (2-Me-Tet-AMPA), (S)-2-amino-3-(3-carboxy-5-methylisoxazol-4-yl)propionic acid (ACPA), and (S)-2-amino-3-(4-bromo-3-hydroxy-isoxazol-5-yl)propionic acid (Br-HIBO), as well as of a mutant thereof (S1S2J-Y702F) in complex with ACPA and Br-HIBO, have been determined. The structures reveal that AMPA agonists with an isoxazole moiety adopt different binding modes in the receptor, dependent on the substituents of the isoxazole. Br-HIBO displays selectivity among different AMPA receptor subunits, and the design and structure determination of the S1S2J-Y702F mutant in complex with Br-HIBO and ACPA have allowed us to explain the molecular mechanism behind this selectivity and to identify key residues for ligand recognition. The agonists induce the same degree of domain closure as AMPA, except for Br-HIBO, which shows a slightly lower degree of domain closure. An excellent correlation between domain closure and efficacy has been obtained from electrophysiology experiments undertaken on non-desensitising GluR2i(Q)-L483Y receptors expressed in oocytes, providing strong evidence that receptor activation occurs as a result of domain closure. The structural results, combined with the functional studies on the full-length receptor, form a powerful platform for the design of new selective agonists.

Two mutants of human heparin binding protein (CAP37): toward the understanding of the nature of lipid A/LPS and BPTI binding.

Heparin binding protein (HBP) is an inactive serine protease homologue with important implications in host defense during infections and inflammations. Two mutants of human HBP, [R23S,F25E]HBP and [G175Q]HBP, have been produced to investigate structure-function relationships of residues in the putative lipid A/lipopolysaccharide (LPS) binding site and BPTI (bovine pancreatic trypsin inhibitor) binding site. The X-ray structures have been determined at 1.9 A resolution for [G175Q]HBP and at 2.5 A resolution for the [R23S,F25E]HBP mutant, and the structures have been fully refined to R-factors of 18.2 % and 20.7 %, respectively. The G175Q mutation does not alter the overall structure of the protein, but the ability to bind BPTI has been eliminated, and the mutant mediates only a limited stimulation of the LPS-induced cytokine release from human monocytes. The lipid A/LPS binding property of [G175Q]HBP is comparable with that of native HBP. The R23S,F25E mutations do not affect the binding of lipid A/LPS and BPTI or the LPS-induced cytokine release from human monocytes. This shows that two diverse ligands, lipid A/LPS and BPTI, do not share binding sites. Previously, there was convincing evidence for the proposed lipid A/LPS binding site of HBP. Unexpectedly, the extensive structural changes introduced by mutation of Arg23 and Phe25 do not affect the binding of lipid A/LPS, indicating that another not yet identified site on HBP is involved in the binding of lipid A/LPS.

Structural basis of cell-cell adhesion by NCAM.

The neural cell adhesion molecule NCAM, a member of the immunoglobulin superfamily, mediates cell-cell recognition and adhesion via a homophilic interaction. NCAM plays a key role during development and regeneration of the nervous system and is involved in synaptic plasticity associated with memory and learning. The 1.85 A crystal structure of the two N-terminal extracellular domains of NCAM reported here provides a structural basis for the homophilic interaction. The molecular packing of the two-domain structure reveals a cross shaped antiparallel dimer, and provides fundamental insight into trans-cellular recognition mediated by NCAM.

Crystallization and molecular-replacement solution of a truncated form of human recombinant tetranectin.

The two C-terminal domains, TN23 (residues 17-181), of human recombinant tetranectin, a plasminogen kringle 4 binding C-type lectin, have been crystallized in two different space groups. Using PEG 8000 as precipitant and at a pH of 8.5, crystals belonging to the monoclinic space group C2 are obtained, with unit-cell parameters a = 160.4, b = 44.7, c = 107.5 A, beta = 127.6 degrees. Using sodium formate as precipitant and at a pH of 5.0, TN23 crystallizes in a rhombohedral space group, with unit-cell parameters a = b = c = 107.4 A, alpha = beta = gamma = 78.3 degrees. A full data set to 4.5 A has been collected from the monoclinic crystals. Using the structure of full-length tetranectin, a molecular-replacement solution has been obtained. The crystal packing shows that TN23 crystallizes as a trimer, with one trimer in the asymmetric unit.

Structure and function of the N-linked glycans of HBP/CAP37/azurocidin: crystal structure determination and biological characterization of nonglycosylated HBP.

The three N-glycosylation sites of human heparin binding protein (HBP) have been mutated to produce a nonglycosylated HBP (ng-HBP) mutant. ng-HBP has been crystallized and tested for biological activity. Complete X-ray data have been collected to 2.1 A resolution, and the structure has been fully refined to an R-factor of 18.4% (R(free) 27.7%). The ng-HBP structure reveals that neither the secondary nor tertiary structure have changed due to the removal of the glycosylation, as compared to the previously determined glycosylated HBP structure. Although the primary events in N-linked glycosylation occurs concomitant with polypeptide synthesis and therefore possesses the ability to influence early events in protein folding, we see no evidence of glycosylation influencing the structure of the protein. The root-mean-square deviation between the superimposed structures was 0.24 A (on C alpha atoms), and only minor local structural differences are observed. Also, the overall stability of the protein seems to be unaffected by glycosylation, as judged by the B-factors derived from the two X-ray structures. The flexibility of a glycan site may be determined by the local polypeptide sequence and structure rather than the glycan itself. The biological in vitro activity assay data show that ng-HBP, contrary to glycosylated HBP, mediates only a very limited stimulation of the lipopolysaccharide induced cytokine release from human monocytes. In animal models of fecal peritonitis, glycosylated HBP treatment rescues mice from and an otherwise lethal injury. It appears that ng-HBP have significant effect on survival, and it can be concluded that ng-HBP can stimulate the host defence machinery albeit to a lesser extent than glycosylated HBP.

Atomic resolution structure of human HBP/CAP37/azurocidin.

Crystals of human heparin binding protein (HBP) diffract to 1.1 A when flash-frozen at 120 K. The atomic resolution structure has been refined anisotropically using SHELXL96. The final model of HBP consists of 221 amino-acid residues of 225 possible, three glycosylation units, one chloride ion, 15 precipitant ethanol molecules and 323 water molecules. The structure is refined to a final crystallographic R factor of 15.9% and Rfree(5%) of 18.9% using all data. A putative protein kinase C activation site has been identified, involving residues 113-120. The structure is compared to the previously determined 2.3 A resolution structure of HBP.

Structure of the C-type lectin carbohydrate recognition domain of human tetranectin.

Tetranectin (TN) is a C-type lectin involved in fibrinolysis, being the only endogenous ligand known to bind specifically to the kringle 4 domain of plasminogen. TN was originally isolated from plasma, but shows a wide tissue distribution. Furthermore, TN has been found in the extracellular matrix of certain human carcinomas, whereas none or little is present in the corresponding normal tissue. The crystal structure of full-length trimeric TN (2.8 A resolution) has recently been published [Nielsen et al. (1997). FEBS Lett. 412, 388-396]. The crystal structure of the carbohydrate recognition domain (CRD) of human TN (TN3) has been determined separately at 2.0 A resolution in order to obtain detailed information on the two calcium binding sites. This information is essential for the elucidation of the specificity of TN towards oligosaccharides. TN3 crystallizes as a dimer, whereas it appears as a monomer in solution. The overall fold of TN3 is similar to other known CRDs. Each monomer is built of two distinct regions, one region consisting of six beta-strands and two alpha-helices, and the other region is composed of four loops harboring two calcium ions. The calcium ion at site 1 forms an eightfold coordinated complex and has Asp116, Glu120, Gly147, Glu150, Asn151, and one water molecule as ligands. The calcium ion at site 2, which is believed to be involved in recognition and binding of oligosaccharides, is sevenfold coordinated with ligands Gln143, Asp145, Glu150, Asp165, and two water molecules. One sulfate ion has been located at the surface of TN3, forming contacts to Glu120, Lys148, Asn106 of a symmetry-related molecule, and to an ethanol molecule.

Crystal structure of tetranectin, a trimeric plasminogen-binding protein with an alpha-helical coiled coil.

Tetranectin is a plasminogen kringle 4-binding protein. The crystal structure has been determined at 2.8 A resolution using molecular replacement. Human tetranectin is a homotrimer forming a triple alpha-helical coiled coil. Each monomer consists of a carbohydrate recognition domain (CRD) connected to a long alpha-helix. Tetranectin has been classified in a distinct group of the C-type lectin superfamily but has structural similarity to the proteins in the group of collectins. Tetranectin has three intramolecular disulfide bridges. Two of these are conserved in the C-type lectin superfamily, whereas the third is present only in long-form CRDs. Tetranectin represents the first structure of a long-form CRD with intact calcium-binding sites. In tetranectin, the third disulfide bridge tethers the CRD to the long helix in the coiled coil. The trimerization of tetranectin as well as the fixation of the CRDs relative to the helices in the coiled coil indicate a demand for high specificity in the recognition and binding of ligands.

Characterization of the allosteric binding pocket of human liver fructose-1,6-bisphosphatase by protein crystallography and inhibitor activity studies.

The structures of three complexes of human fructose-1,6-bisphosphatase (FB) with the allosteric inhibitor AMP and two AMP analogues have been determined and all fully refined. The data used for structure determination were collected at cryogenic temperature (110 K), and with the use of synchrotron radiation. The structures reveal a common mode of binding for AMP and formycine monophosphate (FMP). 5-Amino-4-carboxamido-1 beta-D-5-phosphate-ribofuranosyl-1H-imidazole (AICAR-P) shows an unexpected mode of binding to FB, different from that of the other two ligands. The imidazole ring of AICAR-P is rotated 180 degrees compared to the AMP and FMP bases. This rotation results in a slightly different hydrogen bonding pattern and minor changes in the water structure in the binding pocket. Common features of binding are seen for the ribose and phosphate moieties of all three compounds. Although binding in a different mode, AICAR-P is still capable of making all the important interactions with the residues building the allosteric binding pocket. The IC50 values of AMP, FMP, and AICAR-P were determined to be 1.7, 1.4, and 20.9 microM, respectively. Thus, the approximately 10 times lower potency of AICAR-P is difficult to explain solely from the variations observed in the binding pocket. Only one water molecule in the allosteric binding pocket was found to be conserved in all four subunits in all three structures. This water molecule coordinates to a phosphate oxygen atom and the N7 atom of the AMP molecule, and to similarly situated atoms in the FMP and AICAR-P complexes. This implies an important role of the conserved water molecule in binding of the ligand.

Structure of HBP, a multifunctional protein with a serine proteinase fold.

The structure of human heparin binding protein reveals that the serine proteinase fold has been used as a scaffold for a multifunctional protein with antibacterial activity, monocyte and t-cell activating properties and endotoxin and heparin binding capacity.

X-ray structure of the 154-amino-acid form of recombinant human basic fibroblast growth factor. comparison with the truncated 146-amino-acid form.

The crystal structure of the 154-amino-acid form of human basic fibroblast growth factor (hbFGF154), probably representing the intact form of hbFGF as deduced from the open reading frame of hbFGF cDNA, was determined by X-ray crystallography and refined to a crystallographic residual of 19.0% for all data between 20.0 and 2.0 A resolution. Crystals were obtained from recombinant hbFGF154 expressed in E. coli. hbFGF154 has the same overall structure as the N-terminus truncated 146-amino-acid form. The structure has a Kunitz-type fold and is built of 12 beta-strands of which six antiparallel strands form a beta-sheet barrel. In the structure it was possible to locate two additional residues at the N terminus and the last three C-terminal amino-acid residues, which seem to be disordered in all but one of the reported structures of the truncated form of hbFGF. The C-terminal amino-acid residues are part of the last beta-strand through the formation of a hydrogen bond between the main-chain amide group of Ala152 and the carbonyl O atom of Pro28. An apparent phosphate ion is bound within the basic region on the surface of the molecule and has as ligands the side chains of Asn35, Arg128 and Lys133 and two water molecules. A slightly different hydrogen-bonding pattern to the phosphate ion is observed as compared with the sulfate ions in the truncated forms [Eriksson, Cousens & Matthews (1993). Protein Sci. 2, 1274-1284; Zhang, Cousens, Barr & Sprang (1991). Proc. Natl Acad. Sci. USA, 88, 3446-3450]. One molecule of beta-mercaptoethanol forms a disulfide bridge to Cys77.

Crystal structure of a peptide nucleic acid (PNA) duplex at 1.7 A resolution.

The crystal structure of a PNA duplex reveals both a right- and a left-handed helix in the unit cell. The helices are wide (28A), large pitched (18bp) with the base pairs perpendicular to the helix axis, thereby demonstrating that PNA besides adapting to oligonucleotide partners also has a unique structure by itself.

Human plasminogen binding protein tetranectin: crystallization and preliminary X-ray analysis of the C-type lectin CRD and the full-length protein.

The recombinant human plasminogen binding protein tetranectin (TN) and the C-type lectin CRD of this protein (TN3) have been crystallized. TN3 crystallizes in the tetragonal space group P4(2)2(1)2 with cell dimensions a = b = 64.0, c = 75.7 A and with one molecule per asymmetric unit. The crystals diffract X-rays to at least 2.0 A resolution. A complete diffraction data set has been collected to 2.7 A resolution. The crystals of TN, obtained by the vapour-diffusion reverse salting-in method at 280 K, are rhombohedral, space group R3, with the hexagonal axes a = b = 89.1, c = 75.8 A, and diffract to at least 2.5 A. A full data set has been collected to 3.0 A. The asymmetric unit contains one monomer of TN. Molecular replacement solutions for TN3 and TN have been obtained using the structure of the C-type lectin CRD of rat mannose-binding protein as search model. The rhombohedral space group indicates that trimers of TN are formed in accordance with the observation of trimerization in solution.

Crystallization and molecular replacement solution of human heparin binding protein.

The highly glycosylated protein, human heparin binding protein, has been crystallized in the primitive orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 39.0, b = 66.2 and c = 101.4 A. Ethanol was used as precipitant and glycerol as additive. A full data set has been collected to 3.1 A and diffraction was observed to at least 2.3 A. A molecular replacement solution using human neutrophile elastase as a search model was obtained, showing one molecule per asymmetric unit. The crystal packing showed no bad contacts and the R factor was 44.8% after ten cycles of rigid-body refinement.