Heme packing motifs revealed by the crystal structure of the tetra-heme cytochrome c554 from Nitrosomonas europaea.

Cytochrome c554 (cyt c554), a tetra-heme cytochrome from Nitrosomonas europaea, is an essential component in the biological nitrification pathway. In N. europaea, ammonia is converted to hydroxylamine, which is then oxidized to nitrite by hydroxylamine oxidoreductase (HAO). Cyt c554 functions in the latter process by accepting pairs of electrons from HAO and transferring them to a cytochrome acceptor. The crystal structure of cyt c554 at 2.6 A resolution shows a predominantly alpha-helical protein with four covalently attached hemes. The four hemes are arranged in two pairs such that the planes of the porphyrin rings are almost parallel and overlapping at the edge; corresponding heme arrangements are observed in other multi-heme proteins. Striking structural similarities are evident between the tetra-heme core of cyt c554 and hemes 3-6 of HAO, which suggests an evolutionary relationship between these redox partners.

Structure of the R65Q mutant of yeast 3-phosphoglycerate kinase complexed with Mg-AMP-PNP and 3-phospho-D-glycerate.

The structure of a ternary complex of the R65Q mutant of yeast 3-phosphoglycerate kinase (PGK) with magnesium 5'-adenylylimidodiphosphate (Mg-AMP-PNP) and 3-phospho-D-glycerate (3-PG) has been determined by X-ray crystallography to 2.4 angstrom resolution. The structure was solved by single isomorphous replacement, anamalous scattering, and solvent flattening and has been refined to an R-factor of 0.185, with rms deviations from ideal bond distance and angles of 0.009 angstrom and 1.78 degrees, respectively. PGK consists of two domains, with the 3-PG bound to a "basic patch" of residues from the N-terminal domain and the Mg-AMP-PNP interacting with residues from the C-terminal domain. The two ligands are separated by approximately 11 angstrom across the interdomain cleft. The model of the R65Q mutant of yeast PGK is very similar to the structures of PGK isolated from horse, pig, and Bacillus stearothermophilus (rms deviations between equivalent alpha-carbons in the individual domains < 1.0 angstrom) but exhibits substantial variations with a previously reported yeast structure (rms deviations between equivalent alpha-carbons in the individual domains of 2.9-3.2 angstrom). The most significant tertiary structural differences among the yeast R65Q, equine, porcine, and B. stearothermophilus PGK structures occur in the relative orientations of the two domains. However, the relationships between the observed conformations of PGK are inconsistent with a "hinge-bending" behavior that would close the interdomain cleft. It is proposed that the available structural and biochemical data on PGK may indicate that the basic patch primarily represents the site of anion activation and not the catalytically active binding site for 3-PG.

Crystallization and X-ray structure determination of cytochrome c2 from Rhodobacter sphaeroides in three crystal forms.

Cytochrome c(2) serves as the secondary electron donor that reduces the photo-oxidized bacteriochlorophyll dimer in photosynthetic bacteria. Cytochrome c(2) from Rhodobacter sphaeroides has been crystallized in three different forms. At high ionic strength, crystals of a hexagonal space group (P6(1)22) were obtained, while at low ionic strength, triclinic (P1) and tetragonal (P4(1)2(1)2) crystals were formed. The three-dimensional structures of the cytochrome in all three crystal forms have been determined by X-ray diffraction at resolutions of 2.20 A (hexagonal), 1.95 A, (triclinic) and 1.53 A (tetragonal). The most significant difference observed was the binding of an imidazole molecule to the iron atom of the heme group in the hexagonal structure. This binding displaces the sulfur atom of Met l00, which forms the axial ligand in the triclinic and tetragonal structures.

Structural studies of the binding of the anti-ulcer drug sucrose octasulfate to acidic fibroblast growth factor.

BACKGROUND: The anti-ulcer drug sucrose octasulfate (SOS) binds to fibroblast growth factors (FGFs), proteins which stimulate the growth and differentiation of several cell types, including stomach epithelial cells. It is believed that SOS stabilizes FGFs against acid denaturation in the stomach, thus enhancing their ability to stimulate healing of ulcerated tissue. SOS binds to the same site on FGF as heparin and other proteoglycans; in vivo, FGF must bind to cell-surface proteoglycans or to heparin before it can interact with FGF receptors and stimulate growth. The details of this process are not understood. RESULTS: We report the crystal structure of a 1:1 complex between acidic FGF (aFGF) and SOS at 2.7 A resolution. SOS binds to a positively charged region of aFGF, largely composed of residues 112-127, and makes contacts primarily with Lys112, Arg116, Lys118, and Arg122. This region is also important in binding heparin. The overall conformation of aFGF is not changed by binding SOS, although the positions of some side chains in the binding site shift by as much as 6 A. CONCLUSION: The SOS-FGF crystal structure is consistent with the model that SOS stabilizes FGF by neutralizing several positively charged residues that would destabilize the native structure by electrostatic repulsion. On the basis of this structure, we provide a model for the complex of heparin with an FGF dimer. Such interactions may facilitate FGF receptor dimerization, which may be important in receptor signaling.

X-ray crystal structures of the oxidized and reduced forms of the rubredoxin from the marine hyperthermophilic archaebacterium Pyrococcus furiosus.

The structures of the oxidized and reduced forms of the rubredoxin from the archaebacterium, Pyrococcus furiosus, an organism that grows optimally at 100 degrees C, have been determined by X-ray crystallography to a resolution of 1.8 A. Crystals of this rubredoxin grow in space group P2(1)2(1)2(1) with room temperature cell dimensions a = 34.6 A, b = 35.5 A, and c = 44.4 A. Initial phases were determined by the method of molecular replacement using the oxidized form of the rubredoxin from the mesophilic eubacterium, Clostridium pasteurianum, as a starting model. The oxidized and reduced models of P. furiosus rubredoxin each contain 414 nonhydrogen protein atoms comprising 53 residues. The model of the oxidized form contains 61 solvent H2O oxygen atoms and has been refined with X-PLOR and TNT to a final R = 0.178 with root mean square (rms) deviations from ideality in bond distances and bond angles of 0.014 A and 2.06 degrees, respectively. The model of the reduced form contains 37 solvent H2O oxygen atoms and has been refined to R = 0.193 with rms deviations from ideality in bond lengths of 0.012 A and in bond angles of 1.95 degrees. The overall structure of P. furiosus rubredoxin is similar to the structures of mesophilic rubredoxins, with the exception of a more extensive hydrogen-bonding network in the beta-sheet region and multiple electrostatic interactions (salt bridge, hydrogen bonds) of the Glu 14 side chain with groups on three other residues (the amino-terminal nitrogen of Ala 1; the indole nitrogen of Trp 3; and the amide nitrogen group of Phe 29). The influence of these and other features upon the thermostability of the P. furiosus protein is discussed.

Comparison of the X-ray structure of native rubredoxin from Pyrococcus furiosus with the NMR structure of the zinc-substituted protein.

The three-dimensional X-ray structures of the oxidized and reduced forms of rubredoxin from Pyrococcus furiosus, determined at -161 degrees C, and the NMR structure of the zinc-substituted protein, determined in solution at 45 degrees C, are compared. The NMR and X-ray structures, which were determined independently, are very similar and lead to similar conclusions regarding the interactions that confer hyperthermostability.

Three-dimensional structures of acidic and basic fibroblast growth factors.

Members of the fibroblast growth factor (FGF) family of proteins stimulate the proliferation and differentiation of a variety of cell types through receptor-mediated pathways. The three-dimensional structures of two members of this family, bovine acidic FGF and human basic FGF, have been crystallographically determined. These structures contain 12 antiparallel beta strands organized into a folding pattern with approximate threefold internal symmetry. Topologically equivalent folds have been previously observed for soybean trypsin inhibitor and interleukins-1 beta and -1 alpha. The locations of sequences implicated in receptor and heparin binding by FGF are presented. These sites include beta-sheet strand 10, which is adjacent to the site of an extended sequence insertion in several oncogene proteins of the FGF family, and which shows sequence conservation among the FGF family and interleukin-1 beta.