Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase.

The crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase complexed with a 20-amino acid substrate analog inhibitor has been solved and partially refined at 2.7 A resolution to an R factor of 0.212. The magnesium adenosine triphosphate (MgATP) binding site was located by difference Fourier synthesis. The enzyme structure is bilobal with a deep cleft between the lobes. The cleft is filled by MgATP and a portion of the inhibitor peptide. The smaller lobe, consisting mostly of amino-terminal sequence, is associated with nucleotide binding, and its largely antiparallel beta sheet architecture constitutes an unusual nucleotide binding motif. The larger lobe is dominated by helical structure with a single beta sheet at the domain interface. This lobe is primarily involved in peptide binding and catalysis. Residues 40 through 280 constitute a conserved catalytic core that is shared by more than 100 protein kinases. Most of the invariant amino acids in this conserved catalytic core are clustered at the sites of nucleotide binding and catalysis.

Crystallization and purification of the enzyme anthranilate phosphoribosyl transferase.

Anthranilate phosphoribosyl transferase from the bacterium Hafnia alvei has been crystallized. This enzyme is one of a small number that constitute the biosynthetic pathway for tryptophan. Large cubic crystals were grown at 4 degrees C by dialyzing away the glycerol from a protein solution that included ammonium sulfate, polyethylene glycol and glycerol. The crystals were much more temperature stable and resistant to X-ray deterioration than a previous, similar crystal form that had included glycerol. The crystals belong to the space group I432, a = b = c = 189 A (1 A = 0.1 nm). The ratio of the monomer molecular weight, 37,000, to the volume of the unit cell suggests that there is one homodimer per asymmetric unit. The crystals diffracted to a resolution of 3.0 A at the Stanford Synchotron Radiation Laboratory X-ray source.

Crystallization and preliminary X-ray crystallographic analysis of Mirabilis antiviral protein.

Mirabilis antiviral protein is a single-chain ribosome-inactivating protein purified from the tuberous root of Mirabilis jalapa L. We obtained several forms of crystals of the protein by the hanging drop vapor diffusion method, but most of these crystals were not suitable for X-ray crystallography. After refining the growth conditions, crystals of crystallographic quality were grown in 20-microliters droplets of an equi-volume mixture of 1.5% (w/v) protein solution and a reservoir solution containing 49 to 50% (w/v) ammonium sulfate and 50 mM-ammonium citrate (pH 5.4) at room temperature. Addition of 2 mM-adenine sulfate reduced twinning and "crystal shower". The resulting trigonal crystals diffract beyond 2.5 A resolution using a rotating anode X-ray generator. The space group was determined to be P3(1)21 or P3(2)21 (a = b = 103.9.A, c = 134.6 A, alpha = beta = 90 degrees, gamma = 120 degrees) based on their precession photography of h0l and hk0 zones. There seems to be three monomers in an asymmetric unit for VM = 2.51 A3/Da.

Age differences in perception of risk.

Age differences in perception of risk and the likelihood of disregarding warning signs were examined. A total of 358 adults, between 18 and 85 years old, were asked to rate 12 warning labels with respect to (a) how risky it would be to ignore the warning, (b) how likely it was that they would disregard the warning, and (c) how familiar they were with the labels. The older groups indicated higher risk in ignoring the signs than did the younger group. The older subjects were also less willing to disregard the signs than the younger subjects.

Crystal structures of recombinant human dihydrofolate reductase complexed with folate and 5-deazafolate.

The 2.3-A crystal structure of recombinant human dihydrofolate reductase (EC 1.5.1.3, DHFR) has been solved as a binary complex with folate (a poor substrate at neutral pH) and also as a binary complex with an inhibitor, 5-deazafolate. The inhibitor appears to be protonated at N8 on binding, whereas folate is not. Rotation of the peptide plane joining I7 and V8 from its position in the folate complex permits hydrogen bonding of 5-deazafolate's protonated N8 to the backbone carbonyl of I7, thus contributing to the enzyme's greater affinity for 5-deazafolate than for folate. In this respect it is likely that bound 5-deazafolate furnishes a model for 7,8-dihydrofolate binding and, in addition, resembles the transition state for folate reduction. A hypothetical transition-state model for folate reduction, generated by superposition of the DHFR binary complexes human.5-deazafolate and chicken liver.NADPH, reveals a 1-A overlap of the binding sites for folate's pteridine ring and the dihydronicotinamide ring of NADPH. It is proposed that this binding-site overlap accelerates the reduction of both folate and 7,8-dihydrofolate by simultaneously binding substrate and cofactor with a sub van der Waals separation that is optimal for hydride transfer.

X-ray structures of recombinant yeast cytochrome c peroxidase and three heme-cleft mutants prepared by site-directed mutagenesis.

The 2.2-A X-ray structure for CCP(MI), a plasmid-encoded form of Saccharomyces cerevisiae cytochrome c peroxidase (CCP) expressed in Escherichia coli [Fishel, L.A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360], has been solved, together with the structures of three specifically designed single-site heme-cleft mutants. The structure of CCP(MI) was solved by using molecular replacement methods, since its crystals grow differently from the crystals of CCP isolated from bakers' yeast used previously for structural solution. Small distal-side differences between CCP(MI) and bakers' yeast CCP are observed, presumably due to a strain-specific Thr-53----Ile substitution in CCP(MI). A Trp-51----Phe mutant remains pentacoordinated and exhibits only minor distal structural adjustments. The observation of a vacant sixth coordination site in this structure differs from the results of solution resonance Raman studies, which predict hexacoordinated high-spin iron [Smulevich, G., Mauro, J.M., Fishel, L. A., English, A. M., Kraut, J., & Spiro, T. G. (1988) Biochemistry 27, 5477-5485]. The coordination behavior of this W51F mutant is apparently altered in the presence of a precipitating agent, 30% 2-methyl-2,4-pentanediol. A proximal Trp-191----Phe mutant that has substantially diminished enzyme activity and altered magnetic properties [Mauro, J. M., Fishel, L. F., Hazzard, J. T., Meyer, T. E., Tollin, G., Cusanovich, M. A., & Kraut, J. (1988) Biochemistry 27, 6243-6256] accommodates the substitution by allowing the side chain of Phe-191, together with the segment of backbone to which it is attached, to move toward the heme. This relatively large (ca. 1 A) local perturbation is accompanied by numerous small adjustments resulting in a slight overall compression of the enzyme's proximal domain; however, the iron coordination sphere is essentially unchanged. This structure rules out a major alteration in protein conformation as a reason for the dramatically decreased activity of the W191F mutant. Changing proximal Asp-235 to Asn results in two significant localized structural changes. First, the heme iron moves toward the porphyrin plane, and distal water 595 now clearly resides in the iron coordination sphere at a distance of 2.0 A. The observation of hexacoordinated iron for the D235N mutant is in accord with previous resonance Raman results. Second, the indole side chain of Trp-191 has flipped over as a result of the mutation; the tryptophan N epsilon takes part in a new hydrogen bond with the backbone carbonyl oxygen of Leu-177.(ABSTRACT TRUNCATED AT 400 WORDS)