Biomedical subjects

Frances Jurnak

Publications and source records attributed to Frances Jurnak.

6 recordsLinked to original sources

PubMed2006

Molecular complementarity between tetracycline and the GTPase active site of elongation factor Tu.

Two crystal forms of a complex between trypsin-modified elongation factor Tu-MgGDP from Escherichia coli and the antibiotic tetracycline have been solved by X-ray diffraction analysis to resolutions of 2.8 and 2.1 A, respectively. In the P2(1) form, cocrystals were grown from a solution mixture of the protein and tetracycline. Six copies of the trypsin-modified EF-Tu-MgGDP-tetracycline complex are arranged as three sets of dimers in the asymmetric unit. In the second crystal form, tetracycline was diffused into P4(3)2(1)2 crystals, resulting in a monomeric complex in the asymmetric unit. Atomic coordinates have been refined to crystallographic R factors of 18.0% for the P2(1) form and 20.0% for the P4(3)2(1)2 form. In both complexes, tetracycline makes significant interactions with the GTPase active site of EF-Tu. The phenoldiketone moiety of tetracycline interacts directly with the Mg(2+), the alpha-phosphate group of GDP and two amino acids, Thr25 and Asp80, which are conserved in the GX(4)GKS/T and DX(2)G sequence motifs found in all GTPases and many ATPases. The molecular complementarity, previously unrecognized between invariant groups present in all GTPase/ATPases and the active moiety of tetracycline, may have wide-ranging implications for all drugs containing the phenoldiketone moiety as well as for the design of new compounds targeted against a broad range of GTPases or ATPases.

Binding Sites↗

PubMed2006

Solving the structure of Escherichia coli elongation factor Tu using a twinned data set.

Escherichia coli elongation factor Tu-GDP (EF-Tu-GDP) was crystallized in the presence of novel inhibitors. The only crystals which could be grown were epitaxially as well as merohedrally twinned, highly mosaic and diffracted to a resolution of 3.4 A in space group P3(1)21, with unit-cell parameters a = b = 69.55, c = 169.44 A, alpha = beta = 90, gamma = 120 degrees . To determine whether an inhibitor was present in the crystal, a poor-quality X-ray diffraction data set had to be processed. The three-dimensional structure was ultimately solved and the original question answered. The results also reveal a new type of dimer packing for EF-Tu-GDP.

Anti-Bacterial Agents↗

PubMed2005

Alterations in the expression of the apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/Ref-1) in human melanoma and identification of the therapeutic potential of resveratrol as an APE/Ref-1 inhibitor.

Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/Ref-1) is a multifunctional protein involved in DNA base excision repair and redox regulation of many transcription factors. In different melanoma cell lines, we found that both nucleus and cytoplasm exhibited higher levels of Ref-1 compared with normal melanocytes. Similar increases of Ref-1 expression, detected by immunohistofluorescence, were also evident in nevi and malignant melanoma biopsies compared with normal skin, which were predominantly localized in the nucleus. Using recombinant adenovirus Adref-1, encoding full-length Ref-1, we transiently overexpressed APE/Ref-1 in human melanocytes, which protected these cells from UVB-induced apoptosis and increased foci formation in culture. Ref-1 overexpression also protected melanoma cells from cisplatin- or H2O2-induced apoptosis, whereas increased apoptosis was observed with Ref-1 antisense construct infection. These observations suggested that intracellular Ref-1 levels played an important role in sensitization of melanoma cells to apoptosis. Electrophoretic mobility shift assay results showed that in both cultured primary and metastatic melanomas DNA-binding activities of activator protein-1 and nuclear factor-kappaB were significantly diminished or shifted when anti-APE/Ref-1 antibody was added to deplete APE/Ref-1 from the binding complexes. Induced nuclear factor-kappaB transcriptional activities were also evident after Ref-1 overexpression. Furthermore, using three-dimensional molecular structure modeling and virtual screening, we found that resveratrol, a natural compound found in fruits and vegetables, docks into a druggable pocket of Ref-1 protein. In vitro studies revealed that resveratrol inhibited, in a dose-dependent manner, Ref-1-activated activator protein-1 DNA-binding activities as well as Ref-1 endonuclease activities and rendered melanoma cells more sensitive to dacarbazine treatment.

DNA-(Apurinic or Apyrimidinic Site) Lyase↗

PubMed2003

Resolving the space-group ambiguity of crystals of tomato fruit polygalacturonase.

The first polygalacturonase from a plant, tomato fruit PG2, has been crystallized and data have been collected to a resolution of 1.87 A. The autoindexing program strongly favors one of the primitive orthorhombic cells. A plausible molecular-replacement solution for two molecules in the asymmetric unit has been found for data assigned to space group P2(1)2(1)2(1). Although the numerical criteria and the electron-density maps are reasonable for this solution, manually adjusted models do not refine to an R factor below 0.48. Visual inspection of hkl Bragg planes does not reveal a breakdown in mm symmetry. Nevertheless, the correct space group has been determined to be P2(1), with similar unit-cell parameters, a beta angle of 90.04 degrees and four molecules in the asymmetric unit. The R(sym) of 0.053 for data processed in P2(1)2(1)2(1) is very similar to the R(sym) of 0.047 for the same data processed in P2(1). Comparisons of the intermediate results using the P2(1)2(1)2(1) and P2(1) data sets are provided and the subtle indications of an initial erroneous space-group assignment are discussed.

Crystallization↗

PubMed2003

Characterization and implications of Ca2+ binding to pectate lyase C.

Ca(2+) is essential for in vitro activity of Erwinia chrysanthemi pectate lyase C (PelC). Crystallographic analyses of 11 PelC-Ca(2+) complexes, formed at pH 4.5, 9.5, and 11.2 under varying Ca(2+) concentrations, have been solved and refined at a resolution of 2.2 A. The Ca(2+) site represents a new motif for Ca(2+), consisting primarily of beta-turns and beta-strands. The principal differences between PelC and the PelC-Ca(2+) structures at all pH values are the side-chain conformations of Asp-129 and Glu-166 as well as the occupancies of four water molecules. According to calculations of pK(a) values, the presence of Ca(2+) and associated structural changes lower the pK(a) of Arg-218, the amino acid responsible for proton abstraction during catalysis. The Ca(2+) affinity for PelC is weak, as the K(d) was estimated to be 0.132 (+/-0.004) mm at pH 9.5, 1.09 (+/-0.29) mm at pH 11.2, and 5.84 (+/-0.41) mm at pH 4.5 from x-ray diffraction studies and 0.133 (+/-0.045) mm at pH 9.5 from intrinsic tryptophan fluorescence measurements. Given the pH dependence of Ca(2+) affinity, PelC activity at pH 4.5 has been reexamined. At saturating Ca(2+) concentrations, PelC activity increases 10-fold at pH 4.5 but is less than 1% of maximal activity at pH 9.5. Taken together, the studies suggest that the primary Ca(2+) ion in PelC has multiple functions.

Binding Sites↗

PubMed2003

EF-Tu binding peptides identified, dissected, and affinity optimized by phage display.

The highly abundant GTP binding protein elongation factor Tu (EF-Tu) fulfills multiple roles in bacterial protein biosynthesis. Phage-displayed peptides with high affinity for EF-Tu were selected from a library of approximately 4.7 x 10(11) different peptides. The lack of sequence homology among the identified EF-Tu ligands demonstrates promiscuous peptide binding by EF-Tu. Homolog shotgun scanning of an EF-Tu ligand was used to dissect peptide molecular recognition by EF-Tu. All homolog shotgun scanning selectants bound to EF-Tu with higher affinity than the starting ligand. Thus, homolog shotgun scanning can simultaneously optimize binding affinity and rapidly provide detailed structure activity relationships for multiple side chains of a polypeptide ligand. The reported peptide ligands do not compete for binding to EF-Tu with various antibiotic EF-Tu inhibitors, and could identify an EF-Tu peptide binding site distinct from the antibiotic inhibitory sites.

Amino Acid Sequence↗