Showing papers by "Emil F. Pai published in 1994"
TL;DR: The X-ray structures of the guanine nucleotide binding domains (amino acids 1-166) of five mutants of the H-ras oncogene product p21 were determined and the Gly-12 mutants the larger side chains interfere with GTP binding and/or hydrolysis.
380 citations
TL;DR: The structure of p21 (G12P) is remarkably similar to that of wild-type p21 in the active site, including the position of the nucleophilic water, and the interaction between the carboxylate group of Asp-12 and the gamma-phosphate is mediated by a shared proton, which is shown to exist in solution as well.
Abstract: The three-dimensional structures and biochemical properties of two mutants of the G-domain (residues 1-166) of p21H-ras, p21 (G12D) and p21 (G12P), have been determined in the triphosphate-bound form using guanosine 5'-(beta,gamma-imido)triphosphate (GppNHp). They correspond to the most frequent oncogenic and the only nononcogenic mutation of Gly-12, respectively. The G12D mutation is the only mutant analyzed so far that crystallizes in a space group different from wild type, and the atomic model of the protein shows the most drastic changes of structure around the active site as compared to wild-type p21. This is due to the interactions of the aspartic acid side chain with Tyr-32, Gln-61, and the gamma-phosphate, which result in reduced mobility of these structural elements. The interaction between the carboxylate group of Asp-12 and the gamma-phosphate is mediated by a shared proton, which we show by 31P NMR measurements to exist in solution as well. The structure of p21 (G12P) is remarkably similar to that of wild-type p21 in the active site, including the position of the nucleophilic water. The pyrrolidine ring of Pro-12 points outward and seems to be responsible for the weaker affinity toward GAP (GTPase-activating protein) and the failure of GAP to stimulate GTP hydrolysis.
95 citations
TL;DR: The three‐dimensional structure of trypanothione reductase (TR) from Trypanosoma cruzi has been solved at 0.33 nm resolution by molecular replacement using the structure of C. fasciculata TR as a starting model.
Abstract: The three-dimensional structure of trypanothione reductase (TR) (EC 1.6.4.8) from Trypanosoma cruzi has been solved at 0.33 nm resolution by molecular replacement using the structure of C. fasciculata TR as a starting model. Elucidation of the T. cruzi TR structure represents the first step in the rational design of a drug against Chagas' disease. The structure of T. cruzi TR is compared with those of C. fasciculata TR as well as human and E. coli glutathione reductase (GR). In the FAD-binding domain, TR has two insertions, each about 10 residues long, which do not occur in GR. The first one is a rigid loop stabilizing the position of helix 91–117 which is responsible for the wider active site of TR as compared to GR. The second insertion does not occur where it is predicted by sequence alignment; rather the residues extend three strands of the 4-stranded β-sheet by one or two residues each. This increases the number of hydrogen bonds within the sheet structure. The structure of the NADPH.TR complex has been solved at 0.33 nm resolution. The nicotinamide ring is sandwiched between the flavin ring and the side chain of Phe-198 which undergoes the same conformational change upon coenzyme binding as Tyr-197 in GR. In addition to Arg-222 and Arg-228, which are conserved in TR and GR, Tyr-221—the last residue of the second β-sheet strand of the βαβ dinucleotide binding fold—is in hydrogen bonding distance to the 2′ phosphate group of NADPH. © 1994 John Wiley & Sons, Inc.
65 citations
TL;DR: The refinement of five data sets collected within 2 min at different times after the initiation of the intrinsic GTPase reaction of the protein indicates that the synchrotron Laue method can be used to detect small structural changes and alternative conformations, but is presently limited in the analysis of larger rearrangements since these produce diffuse and broken electron density.
Abstract: The parameters affecting the crystal quality of complexes between p21H-ras and caged GTP have been investigated. The use of pure diastereomers of caged GTP complexed to the more stable p21(G12P)′ mutant of p21 and the addition of n-octyl-β-d-glucopyranoside improved the reproducibility and decreased the mosaicity of the crystals significantly. Furthermore, the crystallization technique was changed from the batch method to the sitting-drop technique. With the availability of a larger yield of well ordered crystals, it was possible to extend the time-resolved crystallographic investigations on p21H-ras. A structure of p21(G12P)′:GTP could be obtained 2 min after photolytic removal of the cage group and led to the identification of a previously unidentified conformation for the so-called catalytically active loop L4. The refinement of five data sets collected within 2 min at different times (2–4, 11–13, 20–22, 30–32 and 90–92 min) after the initiation of the intrinsic GTPase reaction of the protein indicates that the synchrotron Laue method can be used to detect small structural changes and alternative conformations, but is presently limited in the analysis of larger rearrangements since these produce diffuse and broken electron density.
22 citations
TL;DR: Crystallized C-terminally truncated forms of p21(H-ras), with guanosine or GMP bound, in the space groups P4(3)2(1)2, P2( 1)2 (1) 2 and P1(1), and an unexpected electron-density peak was found close to the position of the beta-phosphate in the phosphate-binding loop.
Abstract: p21 is a small guanine nucleotide binding protein that is involved in intracellular signal transduction. Biochemical data suggest that the presence of the beta-phosphate is essential for strong binding of guanine nucleotides to the protein. Guanosine or GMP bind six orders of magnitude more weakly to p21 than GDP or GTP. Moreover, the thermal stability of the protein is dramatically reduced when bound to GMP or guanosine. We have crystallized C-terminally truncated forms of p21(H-ras), with guanosine or GMP bound, in the space groups P4(3)2(1)2, P2(1)2(1)2 and P2(1). The crystals diffract in the range 2.8-2.2 A. Details of the crystallization procedures, the characterization of the crystals and preliminary results of structure determination are described. An unexpected electron-density peak was found close to the position of the beta-phosphate in the phosphate-binding loop.
9 citations
TL;DR: The bifunctional flavoenzyme 5‐hydroxyvaleryl‐CoA dehydratase/ dehydrogenase has been crystallized from solutions containing ammonium sulfate or polyethylene glycol as precipitant and most probably there are two monomers per asymmetric unit.
Abstract: The bifunctional flavoenzyme 5-hydroxyvaleryl-CoA dehydratase/ dehydrogenase has been crystallized from solutions containing ammonium sulfate (form I) or polyethylene glycol (form II) as precipitant. In both cases, the crystals grew in the monoclinic space group C2. The unit cell dimensions for form I crystals were determined as a = 162.8 A, b = 71.8 A, c = 83.5 A, β = 109.1°. Corresponding values for form II crystals were a = 161.2 A, b = 71.6 A, c = 82.2 A, β = 109.3°. In both cases most probably there are two monomers per asymmetric unit. The crystals diffract to about 2 A resolution and are rather stable in the X-ray beam. © 1994 Wiley-Liss, Inc.
3 citations
TL;DR: The tetrameric flavoenzyme 2,4-pentadienoyl-CoA reductase has been crystallized from solutions containing polyethylene glycol as precipitant and complete data sets to about 2.9 A resolution have been collected.
Abstract: The tetrameric flavoenzyme 2,4-pentadienoyl-CoA reductase has been crystallized from solutions containing polyethylene glycol as precipitant. The crystals grow in the monoclinic space group C2 with unit-cell dimensions a = 160.2, b = 120.2, c = 95.3 A, beta = 99.0 degrees. The packing parameter V(M) is 2.3 A(3) Da(-1) (Matthews parameter) for four monomers per asymmetric unit. Complete data sets to about 2.9 A resolution have been collected.
1 citations