Crystallization of Sindbis virus and its nucleocapsid.

Crystals of Sindbis virus, which contains a lipid-bilayer membrane, have been grown using polyethylene glycol. The space group is R32, a = b = 640 A, c = 1520 A. The crystals are highly mosaic, and recorded diffraction is therefore restricted to spacings of about 30 A. The crystals show that the packing of glycoproteins E1 and E2 in the icosahedral outer shell is sufficiently precise that it permits regular and repeated interactions between virus particles in the lattice. Crystals of Sindbis nucleocapsids have also been grown. The limited diffraction data are consistent with close packing of nucleocapsids 404 A in diameter.

Expression and crystallization of a soluble form of Drosophila fasciclin III.

A truncated form of Drosophila fasciclin III has been engineered by site-directed mutagenesis. Secreted fasciclin III is expressed at 35 to 40 mg/l in insect cells with baculovirus carrying the recombinant gene. Single crystals of purified soluble fasciclin III have been grown by vapor diffusion versus polyethylene glycol 8000/sodium citrate at low pH. The space group is P6(1)22 or its enantiomorph P6(5)22, with unit cell dimensions a = b = 140 A, c = 260 A. Cryo-preserved crystals diffract to reciprocal lattice spacings beyond 3.0 A.

Proteolytic dissection of Sindbis virus core protein.

Mild trypsin treatment of the Sindbis virus nucleocapsid protein yields a fragment with a molecular mass of approximately 18.5 kilodaltons with its N terminus at residue 105. The fragment, which is stable to further digestion, appears by gel exclusion chromatography to be monomeric. These data are consistent with a model for the alphavirus core proteins, consisting of an extended and flexible N-terminal arm (residues 1 to 103) and a compactly folded C-terminal domain (residues 104 to 274), as previously suggested on the basis of sequence characteristics.

Manganese and iron superoxide dismutases are structural homologs.

The crystal structure of a tetrameric manganese superoxide dismutase from a thermophilic bacterium, Thermus thermophilus HB8, has been determined at 4.4-A resolution by local averaging of electron density maps calculated by isomorphous replacement. The spatial arrangement of the principal secondary structural features of iron superoxide dismutase is conserved in manganese dismutase. The structural homology is displayed by orienting the polypeptide chain of Escherichia coli Fe dismutase in the electron density map of Mn dismutase. Densities corresponding to bound Mn3+ occur at locations equivalent to the Fe3+ positions in iron dismutase, indicating one metal binding site per chain, or four sites per tetramer. The Mn tetramer, with 222 symmetry, is approximately rectangular in shape and appears to be constructed with only two unique interfaces. One set of interchain contacts closely resembles the dimer interface of Fe dismutase, but the other interface utilizes an inserted polypeptide segment that has no equivalent in Fe dismutase.

The structure of manganese superoxide dismutase from Thermus thermophilus HB8 at 2.4-A resolution.

An atomic model of tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been built into an electron density map at 2.4 A resolution, using chemical sequences of Mn dismutases from Thermus aquaticus and Bacillus stearothermophilus. The monomer fold is structurally very similar to the fold of iron dismutase and comprises two domains, each contributing two ligands to the metal. The Mn(III) ion is bound by protein ligands assigned as His 28, His 83, Asp 165, and His 169. Near neighbors in the metal-ligand environment include a series of hydrophobic residues, Phe 86, Trp 87, Trp 131, and Trp 167. The hydroxyl groups of two Tyr residues, at 36 and 182, are less than 7 A from the metal, as is His 32. Gln 150 forms a bridge between Tyr 36 and Trp 131. These ligands and nearby residues are strongly conserved in the known sequences of Mn dismutases. Only one of the two oxygens of Asp 165 has been assigned as a metal ligand, so that in the current model four protein atoms bind Mn(III). These ligand atoms form part of an approximate trigonal bipyramid in which water may occupy an axial position on the side opposite His 28. The conformation of the protein is unusual in the vicinity of the first ligand, His 28, as a consequence of the insertion of an extra residue in an alpha-helix. The distortion of the helix allows His 32 to stack against the ligand, His 169, and brings Tyr 36 close to the Mn ion. Across one of the dimer interfaces, the two Mn ions are separated by about 18 A, and active center residues from adjoining subunits interdigitate; Tyr 172 interacts with His 32 of the neighboring chain and Glu 168 with the backbone of 168 and with the ligand His 169 from the opposite subunit. Only one other dimer interface occurs in the tetramer; it involves residues 55-62 and sequences near 140 and 156. The center of the oligomeric molecule is filled with solvent.

Expression, purification, crystallization and crystallographic characterization of the human MHC class I related protein MICA.

Crystals of the human MHC-encoded molecule MICA, a homologue of MHC class I proteins, have been grown in hanging-drop vapor-diffusion trials using ammonium sulfate as a precipitating agent with recombinant protein expressed in a baculovirus-based system. Cryo-preserved crystals of MICA belong to the cubic space group F4132 with lattice constants a = b = c = 260.7 A and diffract to a resolution limit of 3.0 A when cryo-preserved. These crystals do not diffract when handled conventionally.

Structural analysis of mutants of high-affinity and low-affinity p-azophenylarsonate-specific antibodies generated by alanine scanning of heavy chain complementarity-determining region 2.

Alanine scanning was used to determine the affinity contributions of 10 side chain amino acids (residues at position 50-60 inclusive) of H chain complementarity-determining region 2 (HCDR2) of the somatically mutated high-affinity anti-p-azophenylarsonate Ab, 36-71. Each mutated H chain gene was expressed in the context of mutated (36-71L) and the unmutated (36-65L) L chains to also assess the contribution of L chain mutations to affinity. Combined data from fluorescence quenching, direct binding, inhibition, and capture assays indicated that mutating H:Tyr(50) and H:Tyr(57) to Ala in the 36-71 H chain results in significant loss of binding with both mutated (36-71L) or unmutated (36-65L) L chain, although the decrease was more pronounced when unmutated L chain was used. All other HCDR2 mutations in 36-71 had minimal effect on Ab affinity when expressed with 36-71 L chain. However, in the context of unmutated L chain, of H:Gly(54) to Ala resulted in significant loss of binding, while Abs containing Asn(52) to Ala, Pro(53) to Ala, or Ile(58) to Ala mutation exhibited 4.3- to 7.1-fold reduced affinities. When alanine scanning was performed instead on certain HCDR2 residues of the germline-encoded (unmutated) 36-65 Ab and expressed with unmutated L chain as Fab in bacteria, these mutants exhibited affinities similar to or slightly higher than the wild-type 36-65. These findings indicate an important role of certain HCDR2 side chain residues on Ab affinity and the constraints imposed by L chain mutations in maintaining Ag binding.

Conservation of binding site geometry among p-azophenylarsonate-specific antibodies.

Murine A/J anti-p-azophenylarsonate mAb that express a dominant cross-reactive Id are encoded by a single set of germ-line VH and VL region genes. The crystal structure of the Fab of antibody 36-71, which uses this canonical set of genes but is somatically mutated, was previously determined. An Fab 36-71:phenylarsonate complex was modeled, identifying amino acid side chains that were proposed as contact residues to hapten. The remarkable conservation of these residues among canonical anti-p-azophenylarsonate antibodies suggested that the overall binding site geometry was maintained among somatically mutated antiarsonate monoclonal antibodies. To test this hypothesis, we used the germ-line-encoded antibody 36-65 to construct mutant antibodies, using oligonucleotide-directed mutagenesis, which differed only at the putative H chain hapten-contacting residues, and measured their hapten binding. A framework residue at H chain position 47 involved in a hydrogen bond network with CDR residues was also mutated. Substitution of several amino acids at each position permitted evaluation of the stereochemical requirements for binding. The results indicate the importance of aromatic stacking of two H chain tyrosine residues against the phenyl ring of the hapten in maintaining affinity, as well as strict complementarity at H chain position 35. The results are consistent with the crystal model of the combining site, and provide further evidence for conservation of the three-dimensional binding site motif among antiarsonate antibodies that bear a dominant heritable ld.