Maria Tordova

Crystal structure of Escherichia coli uracil DNA glycosylase and its complexes with uracil and glycerol: Structure and glycosylase mechanism revisited†

The DNA repair enzyme uracil DNA glycosylase (UDG) catalyzes the hydrolysis of premutagenic uracil residues from single‐stranded or duplex DNA, producing free uracil and abasic DNA. Here we report the high‐resolution crystal structures of free UDG from Escherichia coli strain B (1.60 Å), its complex with uracil (1.50 Å), and a second active‐site complex with glycerol (1.43 Å). These represent the first high‐resolution structures of a prokaryotic UDG to be reported. The overall structure of the E. coli enzyme is more similar to the human UDG than the herpes virus enzyme. Significant differences between the bacterial and viral structures are seen in the side‐chain positions of the putative general‐acid (His187) and base (Asp64), similar to differences previously observed between the viral and human enzymes. In general, the active‐site loop that contains His187 appears preorganized in comparison with the viral and human enzymes, requiring smaller substrate‐induced conformational changes to bring active‐site groups into catalytic position. These structural differences may be related to the large differences in the mechanism of uracil recognition used by the E. coli and viral enzymes. The pH dependence of kcat for wild‐type UDG and the D64N and H187Q mutant enzymes is consistent with general‐base catalysis by Asp64, but provides no evidence for a general‐acid catalyst. The catalytic mechanism of UDG is critically discussed with respect to these results. Proteins 1999;35:13–24. 

Cryosalts: suppression of ice formation in macromolecular crystallography

Quality data collection for macromolecular cryocrystallography requires suppressing the formation of crystalline or microcrystalline ice that may result from flash-freezing crystals. Described here is the use of lithium formate, lithium chloride and other highly soluble salts for forming ice-ring-free aqueous glasses upon cooling from ambient temperature to 100 K. These cryosalts are a new class of cryoprotectants that are shown to be effective with a variety of commonly used crystallization solutions and with proteins crystallized under different conditions. The influence of cryosalts on crystal mosaicity and diffraction resolution is comparable with or superior to traditional organic cryoprotectants.

Crystal structure of the YjeE protein from Haemophilus influenzae: a putative Atpase involved in cell wall synthesis

A hypothetical protein encoded by the gene YjeE of Haemophilus influenzae was selected as part of a structural genomics project for X‐ray analysis to assist with the functional assignment. The protein is considered essential to bacteria because the gene is present in virtually all bacterial genomes but not in those of archaea or eukaryotes. The amino acid sequence shows no homology to other proteins except for the presence of the Walker A motif G‐X‐X‐X‐X‐G‐K‐T that indicates the possibility of a nucleotide‐binding protein. The YjeE protein was cloned, expressed, and the crystal structure determined by the MAD method at 1.7‐Å resolution. The protein has a nucleotide‐binding fold with a four‐stranded parallel β‐sheet flanked by antiparallel β‐strands on each side. The topology of the β‐sheet is unique among P‐loop proteins and has features of different families of enzymes. Crystallization of YjeE in the presence of ATP and Mg2+ resulted in the structure with ADP bound in the P‐loop. The ATPase activity of YjeE was confirmed by kinetic measurements. The distribution of conserved residues suggests that the protein may work as a “molecular switch” triggered by ATP hydrolysis. The phylogenetic pattern of YjeE suggests its involvement in cell wall biosynthesis. Proteins 2002;48:220–226.

Crystal structure of calcium-independent subtilisin BPN' with restored thermal stability folded without the prodomain

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 P212121 with unit cell parameters a = 53.5 Å, b = 60.3 Å, and c = 83.4 Å. 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. Proteins 31:21–32, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Crystal structure of the disulfide‐stabilized Fv fragment of anticancer antibody B1: Conformational influence of an engineered disulfide bond

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 (B1dsFv) crystallizes in space group P6122 with the unit cell parameters a = b = 80.1 Å, and c = 138.1 Å. The crystal structure of the B1dsFv has been determined at 2.1‐Å 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 Å and in bond angle of 2.74°. Comparisons of the B1dsFv structure with known structures of Fv regions of other immunoglobulin fragments shows closely related secondary and tertiary structures. The antigen combining site of B1dsFv is a deep depression 10‐Å wide and 17‐Å 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. Proteins 31:128–138, 1998. Published 1998 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

The 1.30 A resolution structure of the Bacillus subtilis chorismate mutase catalytic homotrimer.

The crystal structure of the Bacillus subtilis chorismate mutase, an enzyme of the aromatic amino acids biosynthetic pathway, was determined to 1.30 A resolution. The structure of the homotrimer was determined by molecular replacement using orthorhombic crystals of space group P2(1)2(1)2(1) with unit-cell parameters a = 52.2, b = 83. 8, c = 86.0 A. The ABC trimer of the monoclinic crystal structure [Chook et al. (1994), J. Mol. Biol. 240, 476-500] was used as the starting model. The final coordinates are composed of three complete polypeptide chains of 127 amino-acid residues. In addition, there are nine sulfate ions, five glycerol molecules and 424 water molecules clearly visible in the structure. This structure was refined with aniosotropic temperature factors, has excellent geometry and a crystallographic R factor of 0.169 with an R(free) of 0.236. The three active sites of the macromolecule are at the subunit interfaces, with residues from two subunits contributing to each site. This orthorhombic crystal form was grown using ammonium sulfate as the precipitant; glycerol was used as a cryoprotectant during data collection. A glycerol molecule and sulfate ion in each of the active sites was found mimicking a transition-state analog. In this structure, the C-terminal tails of the subunits of the trimer are hydrogen bonded to residues of the active site of neighboring trimers in the crystal and thus cross-link the molecules in the crystal lattice.

The three-dimensional structures of two isoforms of nucleoside diphosphate kinase from bovine retina.

The crystal structures of two isoforms of nucleoside diphosphate kinase from bovine retina overexpressed in Escherischia coli have been determined to 2.4 A resolution. Both the isoforms, NBR-A and NBR-B, are hexameric and the fold of the monomer is in agreement with NDP-kinase structures from other biological sources. Although the polypeptide chains of the two isoforms differ by only two residues, they crystallize in different space groups. NBR-A crystallizes in space group P212121 with an entire hexamer in the asymmetric unit, while NBR-B crystallizes in space group P43212 with a trimer in the asymmetric unit. The highly conserved nucleotide-binding site observed in other nucleoside diphosphate kinase structures is also observed here. Both NBR-A and NBR-B were crystallized in the presence of cGMP. The nucleotide is bound with the base in the anti conformation. The NBR-A active site contained both cGMP and GDP each bound at half occupancy. Presumably, NBR-A had retained GDP (or GTP) from the purification process. The NBR-B active site contained only cGMP.