Biomedical subjects

J H Naismith

Publications and source records attributed to J H Naismith.

46 records · Page 3Linked to original sources

PubMed1996

Structural basis of trimannoside recognition by concanavalin A.

Despite the fact that complex saccharides play an important role in many biological recognition processes, molecular level descriptions of protein-carbohydrate interactions are sparse. The legume lectin concanavalin A (con A), from Canavalia ensiformis, specifically recognizes the trimannoside core of many complex glycans. We have determined the crystal structure of a con A-trimannoside complex at 2.3-A resolution now describe the trimannoside interaction with conA. All three sugar residues are in well defined difference electron density. The 1,6-linked mannose residue is bound at the previously reported monosaccharide binding site; the other two sugars bind in an extended cleft formed by residues Tyr-12, Pro-13, Asn-14, Thr-15, and Asp-16. Hydrogen bonds are formed between the protein and all three sugar residues. In particular, the 1,3-linked mannose residue makes a strong hydrogen bond with the main chain of the protein. In addition, a water molecule, which is conserved in other con A structures, plays an important role in anchoring the reducing sugar unit to the protein. The complex is further stabilized by van der Waals interactions. The structure provides a rationale for the high affinity of con A for N-linked glycans.

Carbohydrate Sequence↗

PubMed1995

Crystallographic evidence for dimerization of unliganded tumor necrosis factor receptor.

Activation of the cell surface receptors for tumor necrosis factor (TNF) is effected by the aggregation of cytoplasmic domains that occurs when the extracellular domains of two or three receptors bind to trimeric TNF alpha or TNF beta. The structure of the type I TNF receptor extracellular domain (sTNF-R1), crystallized in the absence of TNF, has now been determined at 2.25-A resolution. The receptor itself is an elongated molecule comprising four disulfide-rich domains in a nearly linear array. Contrary to expectations, the unliganded domains are found to associate into dimers of two distinct types, in which monomers are related by local two-fold axes of symmetry. In one case, the receptors are antiparallel to each other and associate through an interface that overlaps the TNF binding site. If intact receptors were capable of such an association, their cytoplasmic domains would be separated by over 100 A. This interaction could inhibit signaling in the absence of TNF. Parallel dimers are also observed in which the dimer interface is well separated from the TNF binding site. Associations among TNF-bound parallel dimers could cause receptor clustering. Both dimers bury substantial areas of protein surface and are formed by polar and non-polar interactions.

Binding Sites↗

PubMed1994

Refined structure of concanavalin A complexed with methyl alpha-D-mannopyranoside at 2.0 A resolution and comparison with the saccharide-free structure.

The three-dimensional structure of the complex between methyl alpha-D-mannopyranoside and concanavalin A has been refined at 2.0 A resolution. Diffraction data were recorded from a single crystal (space group P2(1)2(1)2(1), a = 123.7, b = 128.6, c = 67.2 A) using synchrotron radiation at a wavelength of 1.488 A. The final model has good geometry and an R factor of 19.9% for 58 871 reflections (82% complete), within the resolution limits of 8 to 2 A, with F > 1.0sigma(F). The asymmetric unit contains four protein subunits arranged as a dimer of dimers with approximate 222 point symmetry. Each monomer binds one saccharide molecule. Each sugar is bound to the protein by hydrogen bonds and van der Waals contacts. Although the four subunits are not crystallographically equivalent, the protein-saccharide interactions are nearly identical in each of the four binding sites. The differences that do occur between the four sites are in the structure of the water network which surrounds each saccharide; these networks are involved in crystal packing. The structure of the complex is compared with a refined saccharide-free concanavalin A structure. The saccharide-free structure is composed of crystallographically identical subunits, again assembled as a dimer of dimers, but with exact 222 symmetry. In the saccharide complex the tetramer association is different in that the monomers tend to separate resulting in fewer intersubunit interactions. The average temperature factor of the mannoside complex is considerably higher than that of the saccharide-free protein. The binding site in the saccharide-free structure is occupied by three ordered water molecules and the side chain of Asp71 from a neighbouring molecule in the crystal. These occupy positions similar to those of the four saccharide hydroxyls which are hydrogen bonded to the site. Superposition of the saccharide-binding site from each structure shows that the major changes on binding involve expulsion of these ordered solvents and the reorientation of the side chain of Tyrl00. Overall the surface accessibility of the saccharide decreases from 370 to 100 A(2) when it binds to the protein. This work builds upon the earlier studies of Derewenda et al. [Derewenda, Yariv, Helliwell, Kalb (Gilboa), Dodson, Papiz, Wan & Campbell (1989). EMBO J. 8, 2198-2193] at 2.9 A resolution, which was the first detailed study of lectin-saccharide interactions.

Journal Article↗

PubMed1994

High-resolution structures of single-metal-substituted concanavalin A: the Co,Ca-protein at 1.6 A and the Ni,Ca-protein at 2.0 A.

The molecular structures of cobalt- and nickel-substituted concanavalin A have been refined at 1.6 and 2.0 A resolution, respectively. Both metal derivatives crystallize in space group I222 with approximate cell dimensions a = 89, b = 87 and c = 63 A and one monomer in the asymmetric unit. The final R factor for Co-substituted concanavalin A is 17.8% for 29 211 reflections with F > 1.0sigma(F) between 8.0 and 1.6 A. For Ni-substituted concanavalin A the final R factor is 15.9% for 16 128 reflections with F > 1.0sigma(F) between 8.0 and 2.0 A resolution. Both structures contain a transition-metal binding site and a calcium-binding site but, unlike Cd-substituted concanavalin A, do not have a third metal-binding site. The Co-substituted concanavalin A structure diffracts to the highest resolution of any concanavalin A structure reported to date. A comparison of the structures of Ni-, Co-, Cd-substituted and native concanavalin A gives an indication of coordinate errors, which is a useful baseline for comparisons with saccharide complexes of concanavalin A described in other work. We also give a detailed account of multiple conformations which were found for five side-chain residues.

Journal Article↗

PubMed1994

Two crystal forms of the extracellular domain of type I tumor necrosis factor receptor.

The soluble extracellular domain of human type I tumor necrosis factor receptor (sTNFrI) is a 161 residue polypeptide found in serum and urine. This domain tightly binds tumor necrosis factors (TNF) alpha and beta and, as part of the whole receptor, initiates the powerful biological effects of TNF. The extracellular domain, typical of other TNF receptor superfamily members, comprises four cysteine-rich motifs. We have obtained two crystal forms of the sTNFrI. One crystal form is grown at pH 3.7 with MgSO4 as the precipitant. These crystals are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 78.5 A, b = 85.5 A and c = 67.5 A. A data set to 2.0 resolution has been collected for these crystals. Tetragonal crystals, space group P4(1)2(1)2 (or P4(3)2(1)2), with unit cell dimensions a = 69.0 A and c = 185.5 A are obtained using methylpentanediol as precipitant at pH 8.5. Data to 2.8 A have been measured from these crystals. It appears that both unit cells may contain two molecules in the asymmetric unit. These crystal structures of sTNFrI may reveal possible conformational differences between receptor localized on the cell surface (high pH), the receptor in the endosomal compartments (low pH) and the receptor in a complex with tumor necrosis factor beta. An accurate structure of the receptor and an understanding of its mechanism will provide a basis for rational drug design.

Binding Sites↗

PubMed1993

Refined structure of cadmium-substituted concanavalin A at 2.0 A resolution.

The three-dimensional structure of cadmium-substituted concanavalin A has been refined using X-PLOR. The R factor on all data between 8 and 2 A is 17.1%. The protein crystallizes in space group I222 with cell dimensions a = 88.7, b = 86.5 and c = 62.5 A and has one protein subunit per asymmetric unit. The final structure contains 237 amino acids, two Cd ions, one Ca ion and 144 water molecules. One Cd ion occupies the transition-metal binding site and the second occupies an additional site, the coordinates of which were first reported by Weinzierl & Kalb [FEBS Lett. (1971), 18, 268-270]. The additional Cd ion is bound with distorted octahedral symmetry and bridges the cleft between the two monomers which form the conventional dimer of concanavalin A. This study provides a detailed analysis of the refined structure of saccharide-free concanavalin A and is the basis for comparison with saccharide complexes reported elsewhere.

Journal Article↗

PubMed1993

Trypanosoma cruzi trypanothione reductase. Crystallization, unit cell dimensions and structure solution.

We have reproducibly crystallized recombinant trypanothione reductase from Trypanosoma cruzi. Yellow tetragonal crystals in the shape of elongated prisms have unit cell dimensions of a = 92.8 A, c = 156.6 A, Laue symmetry of 4/m and are suitable for a detailed structural analysis. Diffraction data to 2.7 A resolution have been recorded using synchrotron radiation at the Daresbury laboratory. The structure has been solved by molecular replacement calculations using this synchrotron data and our previously determined Crithidia fasciculata enzyme structure as a search model. The space group has been identified as P4(3) with a homodimer of approximate molecular mass of 108 kDa in the asymmetric unit. Diffraction beyond 2.5 A has been recorded when large freshly grown crystals are exposed to X-rays. Refinement of the structure is in progress.

Animals↗

PubMed1992

Initiating a crystallographic study of a class II fructose-1,6-bisphosphate aldolase.

We have reproducibly crystallized the metal-dependent Class II fructose-1,6-bisphosphate aldolase from Escherichia coli. Crystals in the shape of truncated hexagonal bipyramids have unit cell dimensions of a = b = 78.4 A, c = 290.6 A and are suitable for a detailed structural analysis. The space group has been identified as P6(1)22 or enantiomorph. Data sets to approximately 2.9 A resolution have been recorded using both the Rigaku R-AXIS IIc image plate area detector coupled to a copper target rotating anode X-ray source and using the MAR image plate systems with synchrotron radiation at the EMBL outstation DESY in Hamburg, and at S.R.S. Daresbury. Diffraction beyond 2.5 A has been observed when large freshly grown crystals are used with the synchrotron beam. A data set to this resolution has been collected. Several putative heavy-atom derivative data sets have also been measured using synchrotron radiation facilities and analysis of these data sets is in progress.

Crystallization↗

PubMedDate not supplied

Seeing double: crystal structures of the type I TNF receptor.

The crystal structure of the extracellular domain of the type I tumor necrosis factor receptor (sTNF-R1) has been determined to 2.25 A at pH 7.5. We have also solved the structure of sTNF-R1 at pH 3.7. sTNF-R1 is an elongated molecule consisting of a linear combination of four cysteine-rich motifs. Interestingly, the crystal structure reveals two distinct dimers of the receptor. One dimer is formed by a parallel arrangement of receptors, the other by an antiparallel arrangement of receptors. In the parallel arrangement of the receptors, the tumor necrosis factor (TNF) binding face of the receptor is completely exposed to solvent. However, in the antiparallel arrangement, the TNF binding face is intimately involved in the dimer interactions. Details of these recognition surfaces are discussed. Both these dimer interactions bury substantial surface area, comprise polar and apolar contact surfaces and have complimentary recognition surfaces. Thus these interactions are typical of genuine protein-protein interactions, rather than crystal packing contacts. These dimers may function to inhibit signal transduction in the absence of TNF or in the case of the parallel dimer, promote clustering of TNF/TNF receptor complexes on the cell surface.

Binding Sites↗

PubMedDate not supplied

Tumor necrosis factor receptor superfamily.

Tumor necrosis factor (TNF) is a powerful cytokine which is involved in the immune and pro-inflammatory response. The TNF receptors (TNF-R1 and TNF-R2) are the sole mediators of TNF signaling. The receptors consist of a disulfide rich domain which recognizes TNF, a transmembrane helix, and a cytoplasmic domain. Signaling occurs when a TNF trimer binds two or three receptors in an extracellular complex which permits aggregation and activation of the cytoplasmic domains. The complex is then endocytosed where it dissociates at low pH. We have now determined the structure of the soluble extracellular domain of TNF-R1 in two crystal forms at pH 3.7 in addition to our earlier report of one form at pH 7.5. One low pH form diffracts to 1.85 A and the entire polypeptide sequence has now been traced for this protein. The C-terminal 20 residues of the protein which were disordered in all previous structures show a different topology and disulfide connectivity to that seen in the remainder of the structure. In all crystal forms, the uncomplexed soluble extracellular domain of the type I TNF-R (sTNF-R1) exists as a dimer. At low pH the dimer buries a large amount of solvent accessible surface (2,900 A2), over 800 A2 greater than the area buried by TNF complexation. This dimer at low pH is different than both dimers observed in our previous pH 7.5 structure of unliganded sTNF-R1. We suggest that the low pH dimer forms during endocytosis and as the dimer completely buries the TNF interaction surface, the dimer would break up the receptor TNF complex. We have identified two distinct structural modules in sTNF-R1, a type A and a type B module. We suggest that these modules are the unit of structural conservation rather than the 6 cysteine subdomain. Although the orientation of these modules with respect to each other is sensitive to crystal packing, complexation, and pH, the modules themselves are structurally well conserved between and within the known sTNF-R1 structures. This modular approach will allow us to build accurate models for all members of the TNF-R superfamily.

Animals↗