The low ionic strength crystal structure of horse cytochrome c at 2.1 A resolution and comparison with its high ionic strength counterpart.

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.

MAD analysis of FHIT, a putative human tumor suppressor from the HIT protein family.

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.

Characterization of two crystal forms of neutrophil cationic protein NP2, a naturally occurring broad-spectrum antimicrobial agent from leukocytes.

A hexagonal crystal form (P6(3)22, a = b = 34.0 A, c = 113.5 A) and a monoclinic form (P2(1), a = 37.1 A, b = 32.2 A, c = 32.4 A, beta = 110 degrees) of neutrophil cationic protein NP2, isolated from rabbit leukocytes, have been characterized. The monoclinic form, containing two promoters (Mr = 3844) per asymmetric unit, diffracts to at least 1.8 A and is suitable for high-resolution structural studies.

The 2.4 A crystal structure of cholera toxin B subunit pentamer: choleragenoid.

Cholera toxin, a heterohexameric AB5 enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts. Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding the GM1 gangliosides exposed on the luminal surface of intestinal epithelial cells. The crystal structure of choleragenoid has been independently solved and refined at 2.4 A resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging. The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin, choleragen, the heat-labile enterotoxin from Escherichia coli, and for a choleragenoid-GM1 pentasaccharide complex. In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel. The binding of choleragenoid to the A subunit or to its receptor pentasaccharide modestly affects the local stereochemistry without perceptibly altering the subunit interface.

The three-dimensional crystal structure of cholera toxin.

The clinical manifestations of cholera are largely attributable to the actions of a secreted hexameric AB5 enterotoxin (choleragen). We have independently solved and refined the three-dimensional structure of choleragen at 2.5 A resolution. The structure of the crystalline toxin closely resembles that described for the heat-labile enterotoxin from Escherichia coli (LT) with which it shares 80% sequence homology. In both cases, the wedge-shaped A subunit is loosely held high above the plane of the pentameric B subunits by the tethering A2 chain. The most striking difference between the two toxins occurs at the carboxyl terminus of the A2 chain. Whereas the last 14 residues of the A2 chain of LT threading through the central pore of the B5 assembly form an extended chain with a terminal loop, the A2 chain of choleragen remains a nearly continuous alpha-helix throughout its length. The four carboxyl-terminal residues of the A2 chain (KDEL sequence), disordered in the crystal structure of LT, are clearly visible in choleragen's electron-density map. In the accompanying article we describe the three-dimensional structure of the isolated B pentamer of cholera toxin (choleragenoid). Comparison of the crystalline coordinates of choleragen, choleragenoid, and LT provides a solid three-dimensional foundation for further experimental investigation. These structures, along with those of related toxins from Shigella dysenteria and Bordetella pertussis, offer a first step towards the rational design of new vaccines and anti-microbial agents.

Purification, crystallization and preliminary crystallographic analysis of porcine aldose reductase.

Large crystals of porcine aldose reductase have been grown from polyethylene glycol solutions. The crystals are triclinic, space-group P1, with a = 81.3 A, b = 85.9 A, c = 56.6 A, alpha = 102.3 degrees, beta = 103.3 degrees and gamma = 79.0 degrees. The crystals grow within ten days to dimensions of 0.6 mm x 0.4 mm x 0.2 mm and diffract to at least 2.5 A. There are four molecules in the unit cell related by a set of three mutually perpendicular non-crystallographic 2-fold axes.

Crystallization and preliminary X-ray diffraction studies of the human adenovirus serotype 2 proteinase with peptide cofactor.

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.

Crystallization of isoelectrically homogeneous cholera toxin.

Past difficulty in growing good crystals of cholera toxin has prevented the study of the crystal structure of this important protein. We have determined that failure of cholera toxin to crystallize well has been due to its heterogeneity. We have now succeeded in overcoming the problem by isolating a single isoelectric variant of this oligomeric protein (one A subunit and five B subunits). Cholera toxin purified by our procedure readily forms large single crystals. The crystal form (space group P2(1), a = 73.0 A, b = 92.2 A, c = 60.6 A, beta = 106.4 degrees, one molecule in the asymmetric unit) has been described previously [Sigler et al. (1977) Science (Washington, D.C.) 197, 1277-1278]. We have recorded data from native crystals of cholera toxin to 3.0-A resolution with our electronic area detectors. With these data, we have found the orientation of a 5-fold symmetry axis within these crystals, perpendicular to the screw dyad of the crystal. We are now determining the crystal structure of cholera toxin by a combination of multiple heavy-atom isomorphous replacement and density modification techniques, making use of rotational 5-fold averaging of the B subunits.

Enzymatic function in crystals of delta 5-3-ketosteroid isomerase. Catalytic activity and binding of competitive inhibitors.

Crystals of the steroid-metabolizing enzyme, delta 5-3-ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni, exhibit many enzymatic properties. Each enzyme subunit in the lattice binds a competitive inhibitor, progesterone, with the same stoichiometry (1:1) and affinity (KD = 6 X 10(-6) M) as the enzyme in solution. Another competitive inhibitor, 19-nortestosterone, competes with progesterone for the same binding sites in the crystal. The enzyme crystals catalyze the conversion of delta 5- to delta 4-ketosteroids, but because the enzyme is so efficient, and substrate diffusion into the crystal is so slow, substrate cannot penetrate deeply into the crystal before being converted to product. A general theoretical formulation is presented to account for the effects of substrate diffusion into enzyme crystals of different shapes and sizes. The dependence of apparent mean enzyme activity in steroid isomerase crystals as a function of crystal size is shown to be consistent with this theoretical formulation. These inhibitor binding and catalytic properties suggest that the enzyme is in an active conformation within these crystals.

Characterization of two crystal forms of human defensin neutrophil cationic peptide 1, a naturally occurring antimicrobial peptide of leukocytes.

Two orthorhombic crystal forms (P2(1)2(1)2, a = 30.5 A, b = 44.5 A, c = 40.5 A; I2(1)2(1)2(1) (or I222), a = 30.1 A, b = 66.5 A, c = 35.5 A) of human neutrophil cationic peptide 1 have been characterized. The P2(1)2(1)2 form contains two peptides (Mr = 3425) per asymmetric unit; the I2(1)2(1)2(1) form contains one peptide per asymmetric unit. Both crystal forms diffract to beyond 1.8 A resolution.

Crystallization and preliminary X-ray crystallographic analysis of FlhD/FlhC complex from Escherichia coli.

The heterotetrameric (C(2)D(2)) FlhD/FlhC complex was first discovered as a transcriptional activator of the flagellar genes in Escherichia coli. Recent studies now show that FlhD/FlhC also regulates several non-flagellar target genes in E. coli. The FlhD/FlhC complex also plays several important roles in other microorganisms. The molecular interactions between FlhD and FlhC, as well as the mechanisms by which the complex may vary its DNA-binding specificity, are not clear. Determination of the FlhD/FlhC crystal structure will provide insight into these protein-protein and protein-DNA interactions. The initial steps in this investigation are reported here: the overexpression, purification and crystallization of the FlhD/FlhC complex, the characterization of this crystal form and the recording and processing of an initial diffraction data set. The obtained crystal form of the FlhD/FlhC complex is hexagonal (space group P6(1), unit-cell parameters a = b = 150.5, c = 115.9 A). The crystal density is very low (V(M) = 5.5), with 81.7% of its volume occupied by solvent. A single C(2)D(2) tetramer is present in the crystallographic asymmetric unit. A complete native data set has been collected to 4.5 A resolution.

The 6-A crystal structure of delta 5-3-ketosteroid isomerase. Architecture and location of the active center.

The crystal structure of the dimeric steroid metabolizing enzyme, delta 5-3-ketosteroid isomerase (EC 5.3.3.1), has been solved to 6-A resolution by multiple isomorphous replacement, augmented by real space direct methods. The unit cell is hexagonal (space group P6122) with dimensions a = b = 65.4 A, c = 504 A, and contains four identical 13,400-dalton protomers in each of its 12 asymmetric units. The 504-A c axis required double focusing mirrors (Franks optics) to resolve the reflections. The complexity of the combined local and lattice symmetry necessitated direct methods to establish the positions of heavy atoms in even the simplest of the isomorphous derivatives. The electron density map clearly showed both (a) the elaborate packing scheme of protomers, which accounts for this large and complicated unit cell, and (b) the coarse features of the functional dimer. The steroid-binding site has been established by imaging the bound inhibitor, 4-acetoxymercuriestradiol, in a difference Fourier map. Each of the dimer's two steroid-binding sites lies completely within one subunit but close enough to the opposing subunit that functional interactions may be possible.

On the number of steroid-binding sites of delta 5-3-oxosteroid isomerase.

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.

Crystallization of tuna ferricytochrome c at low ionic strength.

Previous crystallographic studies of tuna ferricytochrome c have employed crystals grown from solutions of ammonium sulfate, corresponding to an ionic strength of 9.5 M (Takano, T., and Dickerson, R. E. (1981) J. Mol. Biol. 153, 95-115). To obtain a structure at a lower ionic strength, the ferric tuna protein was crystallized at neutral pH with polyethylene glycol at an ionic strength of 45 mM, These crystals (space group P2(1), a = 37.11 A, b = 107.66 A, c = 55.75 A, beta = 105.3 degrees) contain four molecules/asymmetric unit and grow to dimensions of 0.2 X 0.4 X 1.0 mm in 2-4 weeks. They diffract to beyond 1.8 A and are stable in the x-ray beam. We have recorded 28,198 unique Bragg reflections (83% of those possible) to a resolution of 1.89 A from a native crystal. We are undertaking a solution of this structure by the molecular replacement method.

Crystal structure of the global regulator FlhD from Escherichia coli at 1.8 A resolution.

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.

Positioning of a spin-labeled substrate analogue into the structure of delta 5-3-ketosteroid isomerase by combined kinetic, magnetic resonance, and X-ray diffraction methods.

We have shown by kinetic and magnetic resonance measurements that a spin-labeled substrate analogue, spiro[doxyl-2,3'-5' alpha-androstan]-17'beta-ol, binds at the substrate site of crystalline delta 5-3-ketosteroid isomerase (steroid delta-isomerase; EC 5.3.3.1) of Pseudomonas testosteroni. The spin-labeled steroid is a linear competitive inhibitor with a Ki value (25 +/- 5 microM) that is consistent with dissociation constants obtained by direct binding measurements based on changes in the electron paramagnetic resonance spectrum of the nitroxide, longitudinal relaxation rates of water protons, and longitudinal and transverse relaxation rates of carbon-bound protons of the isomerase. These binding studies yield a stoichiometry for the nitroxide of 1 per subunit of the enzyme. Measurements of the longitudinal relaxation rates of water protons indicate that the 3-doxyl portion of the spin-label is highly immobilized yet is exposed to solvent. Paramagnetic effects of the nitroxide on T1 defined distances to several previously assigned [Benisek, W. F., & Ogez, J. R. (1982) Biochemistry 21, 5816-5825] and newly assigned protons of the enzyme. These distances were then used to locate (with an accuracy of +/- 2 A) the nitroxide moiety at a unique position in a partially refined 2.5-A resolution X-ray structure of native isomerase. Three of five additional proton resonance peaks, attributed to ring-shielded methyl groups, could be assigned to specific residues on the basis of distances from the spin-label in the X-ray structure. The remaining portion of the spin-labeled steroid was then docked into the X-ray structure in a hydrophobic cavity of the enzyme. This position of the steroid is consistent with the steroid binding site previously proposed [Westbrook, E. M., Piro, O. E., & Sigler, P. B. (1984) J. Biol. Chem. 259, 9096-9103]. However, the rotational orientation of this steroid about its long axis could not be unambiguously established. If we assume that steroid substrates and the spin-labeled inhibitor bind to the same site, but with reversal of the 3- and 17-positions, then the phenolic hydroxyl of Tyr-55 is optimally positioned to function as the general acid that protonates the 3-keto group of the substrate, facilitated by the negative end of the dipole of a 10-residue alpha-helix, the only helix in the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)