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FlhD is a 13.3 kDa transcriptional activator protein of flagellar genes and a global regulator. FlhD activates the transcription of class II operons in the flagellar regulon when complexed with a second protein FlhC (21.5 kDa). FlhD also regulates other expression systems in Escherichia coli. We are seeking to understand this plasticity of FlhD's DNA-binding specificity and, to this end, we have determined the crystal structure of the isolated FlhD protein. The structure was solved by substituting seleno-methionine for natural sulphur-methionine in FlhD, crystallizing the protein and determining the structure factor phases by the method of multiple-energy anomalous dispersion (MAD). The FlhD protein is dimeric. The dimer is tightly coupled, with an intimate contact surface, implying that the dimer does not easily dissociate. The FlhD monomer is predominantly alpha-helical. The C-termini of both FlhD monomers (residues 83-116) are completely disrupted by crystal packing, implying that this region of FlhD is highly flexible. However, part of the C-terminus structure in chain A (residues 83-98) was modelled using a native FlhD crystal. What is seen in chain A suggests a classic DNA-binding, helix-turn-helix (HTH) motif. FlhD does not bind DNA by itself, so it may be that the DNA-binding HTH motif becomes rigidly defined only when FlhD forms a complex with some other protein, such as FlhC. If this were true, it might explain how FlhD exhibits plasticity in its DNA-binding specificity, as each partner protein with which it forms a complex could allosterically affect the binding specificity of its HTH motif. A disulphide bridge is seen between the unique cysteine residues (Cys-65) of FlhD native homodimers. Alanine substitution at Cys-65 does not affect FlhD transcription activator activity, suggesting that the disulphide bond is not necessary for either dimer stability or this function of FlhD. Electrostatic potential analysis indicates that dimeric FlhD has a negatively charged surface.

Background: The fragile histidine triad (FHIT) protein is a member of the large and ubiquitous histidine triad (HIT) family of proteins. It is expressed from a gene located at a fragile site on human chromosome 3, which is commonly disrupted in association with certain cancers. On the basis of the genetic evidence, it has been postulated that the FHIT protein may function as a tumor suppressor, implying a role for the FHIT protein in carcinogenesis. The FHIT protein has dinucleoside polyphosphate hydrolase activity in vitro, thus suggesting that its role in vivo may involve the hydrolysis of a phosphoanhydride bond. The structural analysis of FHIT will identify critical residues involved in substrate binding and catalysis, and will provide insights into the in vivo function of HIT proteins., Results: The three-dimensional crystal structures of free and nucleoside complexed FHIT have been determined from multiwavelength anomalous diffraction (MAD) data, and they represent some of the first successful structures to be measured with undulator radiation at the Advanced Photon Source. The structures of FHIT reveal that this protein exists as an intimate homodimer, which is based on a core structure observed previously in another human HIT homolog, protein kinase C interacting protein (PKCI), but has distinctive elaborations at both the N and C termini. Conserved residues within the HIT family, which are involved in the interactions of the proteins with nucleoside and phosphate groups, appear to be relevant for the catalytic activity of this protein., Conclusions: The structure of FHIT, a divergent HIT protein family member, in complex with a nucleotide analog suggests a metal-independent catalytic mechanism for the HIT family of proteins. A structural comparison of FHIT with PKCI and galactose-1-phosphate uridylyltransferase (GaIT) reveals additional implications for the structural and functional evolution of the ubiquitous HIT family of proteins.

Recombinant human adenovirus serotype 2 proteinase (both native and selenomethionine-substituted) has been crystallized in the presence of the serotype 12, 11-residue peptide cofactor. The crystals (space group P3(1)21 or P3(2)21, one molecule per asymmetric unit, a = b = 41.3 angstrum, c = 197.0 angstrum) grew in solutions containing 20-40% 2-methyl-2,4-pentanediol (MPD), 0.1-0.2 M sodium citrate, and 0.1 M sodium HEPES, pH 5.0-7.5. Diffraction data (84% complete to 2.2 angstrum resolution with Rmerge of 0.0335) have been measured from cryopreserved native enzyme crystals with the Argonne blue (1,024 x 1,024 pixel array) charge-coupled device detector at beamline X8C at the National Synchrotron Light Source (operated by Argonne National Laboratory's Structural Biology Center). Additionally, diffraction data from selenomethionine-substituted proteinase, 65% complete to 2.0 angstrum resolution with Rmerge values ranging 0.05-0.07, have been collected at three X-ray energies at and near the selenium absorption edge. We have determined three of the six selenium sites and are initiating a structure solution by the method of multiwavelength anomalous diffraction phasing.

Background: Cytochrome c is an integral part of the mitochondrial respiratory chain. It is confined to the intermembrane space of mitochondria, and has the function of transferring electrons between its redox partners. Solution studies of cytochrome c indicate that the conformation of the molecule is sensitive to the ionic strength of the medium., Results: The crystal structures of cytochromes c from several species have been solved at extremely high ionic strengths of near-saturated solutions of ammonium sulfate. Here we present the first crystal structure of ferricytochrome c at low ionic strength refined at 2.1 A resolution. In general, the structure has the same features as those determined earlier. However, there are some differences in both backbone and side-chain conformations in several areas. These areas coincide with those observed by NMR and resonance Raman spectroscopy to be sensitive to ionic strength., Conclusions: Neither ionic strength nor crystal-packing interactions have much influence on the conformation of horse cytochrome c. Nevertheless, some differences in the side-chain conformations at high and low ionic strengths may be important for understanding how the protein functions. Close examination of the gamma-turn (residues 27-29) conserved in cytochromes c leads us to propose the 'negative classical' gamma-turn to describe this unusual feature.

The number of steroid-binding sites in delta 5-3-oxosteroid isomerase of Pseudomonas testosteroni (EC 5.3.3.1) has been determined from measurements of the red shift of the ultraviolet chromophore of 19-nortestosterone upon binding to the enzyme. The experiments include spectroscopic measurements when limiting concentrations of either 19-nortestosterone or isomerase are titrated with varying concentrations of the complementary ligand. Analysis of the results indicates one binding site per subunit (Mr 13 394). Scatchard plots indicate a single family of equivalent binding sites. 5, 10-Secoestr-5-yne-3, 10, 17-trione is a suicide substrate of isomerase [Batzold & Robinson (1975) J. Am. Chem. Soc. 97, 2576]. The time course for inactivation of isomerase with an excess of 5, 10-seco[7-3H]estr-5-yne-3, 10, 17-trione was parallel to the covalent incorporation of steroid and gave a final stoichiometry of nearly one steroid molecule per subunit of enzyme. Alkylation of [14C] isomerase with excess of this 3H-labeled steroid followed by gel-filtration and dialysis gave an inactivated enzyme with a 3H/14C ratio that corresponds to one molecule of steroid bound per subunit; this stoichiometry was constant over a wide range of protein concentrations (0.1--10 mg/ml). Diffusion of [3H]progesterone into hexagonal crystals of isomerase showed that at saturation one steroid molecule was bound per protomer. Taken together these findings strongly support the conclusion that one molecule of steroid is bound per subunit of isomerase both in solution and in the crystal state.