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The biochemical and structural properties of bovine retinal nucleoside diphosphate kinase were investigated. The enzyme showed two polypeptides of approximately 17.5 and 18.5 kDa on SDS-PAGE, while isoelectric focusing revealed seven to eight proteins with a pI range of 7.4-8.2. Sedimentation equilibrium yielded a molecular mass of 96 +/- 2 kDa for the enzyme. Carbohydrate analysis revealed that both polypeptides contained Gal, Man, GlcNAc, Fuc, and GalNac saccharides. Like other nucleoside diphosphate kinases, the retinal enzyme showed substantial differences in the Km values for various di- and triphosphate nucleotides. Immunogold labeling of bovine retina revealed that the enzyme is localized on both the membranes and in the cytoplasm. Screening of a retinal cDNA library yielded full-length clones encoding two distinct isoforms (NBR-A and NBR-B). Both isoforms were overexpressed in Escherichia coli and their biochemical properties compared with retinal NDP-kinase. The structures of NBR-A and NBR-B were determined by X-ray crystallography in the presence of guanine nucleotide(s). Both isoforms are hexameric, and the fold of the monomer is similar to other nucleoside diphosphate kinase structures. The NBR-A active site contained both a cGMP and a GDP molecule each bound at half occupancy while the NBR-B active site contained only cGMP.

The crystal structure of unliganded mutant R111M of human cellular retinoic acid-binding protein type II (apo-CRABPII (R111M)) has been determined at 2.3 A and refined to a crystallographic R-factor of 0. 18. Although the mutant protein has lower affinity for all-trans-retinoic acid (RA) than the wild-type, it is properly folded, and its conformation is very similar to the wild-type. apo-CRABPII (R111M) crystallizes in space group P1 with two molecules in the unit cell. The two molecules have high structural similarity except that their alpha2 helices differ strikingly. Analyses of the molecular conformation and crystal packing environment suggest that one of the two molecules assumes a conformation compatible with RA entry. Three structural elements encompassing the opening of the binding pocket exhibit large conformational changes, when compared with holo-CRABPII, which include the alpha2 helix and the betaC-betaD and betaE-betaF hairpin loops. The alpha2 helix is unwound at its N terminus, which appears to be essential for the opening of the RA-binding pocket. Three arginine side-chains (29, 59, and 132) are found with their guanidino groups pointing into the RA-binding pocket. A three-step mechanism of RA entry has been proposed, addressing the opening of the RA entrance, the electrostatic potential that directs entry of RA into the binding pocket, and the intramolecular interactions that stabilize the RA.CRABPII complex via locking the three flexible structural elements when RA is bound., (Copyright 1998 Academic Press Limited.)

A recombinant Fv construct of the B1 monoclonal antibody that recognizes the LewisY-related carbohydrate epitope on human carcinoma cells has been prepared. The Fv is composed of the polypeptide chains of the VH and VL domains expressed independently and isolated as inclusion bodies. The Fv is prepared by combining and refolding equimolar amounts of guanidine chloride solubilized inclusion bodies. The Fv is stabilized by an engineered interchain disulfide bridge between residues VL100 and VH44. This construct has a similar binding affinity as that of the single-chain construct (Benhar and Pastan, Clin. Cancer Res. 1:1023-1029, 1995). The B1 disulfide-stabilized Fv (BldsFv) crystallizes in space group P6(1)22 with the unit cell parameters a = b = 80.1 A, and c = 138.1 A. The crystal structure of the BldsFv has been determined at 2.1-A resolution using the molecular replacement technique. The final structure has a crystallographic R-value of 0.187 with a root mean square deviation in bond distance of 0.014 A and in bond angle of 2.74 degrees. Comparisons of the BldsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of BldsFv is a deep depression 10-A wide and 17-A long with the walls of the depression composed of residues, many of which are tyrosines, from complementarity determining regions L1, L3, H1, H2, and H3. Model building studies indicate that the LewisY tetrasaccharide, Fuc-Gal-Nag-Fuc, can be accommodated in the antigen combining site in a manner consistent with the epitope predicted in earlier biochemical studies (Pastan, Lovelace, Gallo, Rutherford, Magnani, and Willingham, Cancer Res. 51:3781-3787, 1991). Thus, the engineered disulfide bridge appears to cause little, if any, distortion in the Fv structure, making it an effective substitute for the B1 Fab.

The three-dimensional structure of a subtilisin BPN' construct that was produced and folded without its prodomain shows the tertiary structure is nearly identical to the wild-type enzyme and not a folding intermediate. The subtilisin BPN' variant, Sbt70, was cloned and expressed in Escherichia coli without the prodomain, the 77-residue N-terminal domain that catalyzes the folding of the enzyme into its native tertiary structure. Sbt70 has the high-affinity calcium-binding loop, residues 75 to 83, deleted. Such calcium-independent forms of subtilisin BPN' refold independently while retaining high levels of activity [Bryan et al., Biochemistry, 31:4937-4945, 1992]. Sbt70 has, in addition, seven stabilizing mutations, K43N, M50F, A73L, Q206V, Y217K, N218S, Q271E, and the active site serine has been replaced with alanine to prevent autolysis. The purified Sbt70 folded spontaneously without the prodomain and crystallized at room temperature. Crystals of Sbt70 belong to space group P2(1)2(1)2(1) with unit cell parameters a = 53.5 A, b = 60.3 A, and c = 83.4 A. Comparison of the refined structure with other high-resolution structures of subtilisin BPN' establishes that the conformation of Sbt70 is essentially the same as that previously determined for other calcium-independent forms and that of other wild-type subtilisin BPN' structures, all folded in the presence of the prodomain. These findings confirm the results of previous solution studies that showed subtilisin BPN' can be refolded into a native conformation without the presence of the prodomain [Bryan et al., Biochemistry 31:4937-4945, 1992]. The structure analysis also provides the first descriptions of four stabilizing mutations, K43N, A73L, Q206V, and Q271E, and provides details of the interaction between the enzyme and the Ala-Leu-Ala-Leu tetrapeptide found in the active-site cleft.