Claudine Cohen-Addad

Crystal structure of Pseudomonas fluorescens 4-hydroxyphenylpyruvate dioxygenase: an enzyme involved in the tyrosine degradation pathway

BACKGROUND In plants and photosynthetic bacteria, the tyrosine degradation pathway is crucial because homogentisate, a tyrosine degradation product, is a precursor for the biosynthesis of photosynthetic pigments, such as quinones or tocophenols. Homogentisate biosynthesis includes a decarboxylation step, a dioxygenation and a rearrangement of the pyruvate sidechain. This complex reaction is carried out by a single enzyme, the 4-hydroxyphenylpyruvate dioxygenase (HPPD), a non-heme iron dependent enzyme that is active as a homotetramer in bacteria and as a homodimer in plants. Moreover, in humans, a HPPD deficiency is found to be related to tyrosinemia, a rare hereditary disorder of tyrosine catabolism. RESULTS We report here the crystal structure of Pseudomonas fluorescens HPPD refined to 2.4 A resolution (Rfree 27.6%; R factor 21.9%). The general topology of the protein comprises two barrel-shaped domains and is similar to the structures of Pseudomonas 2,3-dihydroxybiphenyl dioxygenase (DHBD) and Pseudomonas putida catechol 2,3-dioxygenase (MPC). Each structural domain contains two repeated betaalpha betabeta betaalpha modules. There is one non-heme iron atom per monomer liganded to the sidechains of His161, His240, Glu322 and one acetate molecule. CONCLUSIONS The analysis of the HPPD structure and its superposition with the structures of DHBD and MPC highlight some important differences in the active sites of these enzymes. These comparisons also suggest that the pyruvate part of the HPPD substrate (4-hydroxyphenylpyruvate) and the O2 molecule would occupy the three free coordination sites of the catalytic iron atom. This substrate-enzyme model will aid the design of new inhibitors of the homogentisate biosynthesis reaction.

Effects of p47phox C Terminus Phosphorylations on Binding Interactions with p40phox and p67phox STRUCTURAL AND FUNCTIONAL COMPARISON OF p40phox and p67phox SH3 DOMAINS

The neutrophil NADPH oxidase produces superoxide anions in response to infection. This reaction is activated by association of cytosolic factors, p47phox and p67phox, and a small G protein Rac with the membranous flavocytochrome b558. Another cytosolic factor, p40phox, is associated to the complex and is reported to play regulatory roles. Initiation of the NADPH oxidase activation cascade has been reported as consecutive to phosphorylation on serines 359/370 and 379 of the p47phox C terminus. These serines surround a polyproline motif that can interact with the Src homology 3 (SH3) module of p40phox (SH3p40) or the C-terminal SH3 of p67phox (C-SH3p67). The latter one presents a higher affinity in the resting state for p47phox. A change in SH3 binding preference following phosphorylation has been postulated earlier. Here we report the crystal structures of SH3p40 alone or in complex with a 12-residue proline-rich region of p47phox at 1.46 Å resolution. Using intrinsic tryptophan fluorescence measurements, we compared the affinity of the strict polyproline motif and the whole C terminus peptide with both SH3p40 and C-SH3p67. These data reveal that SH3p40 can interact with a consensus polyproline motif but also with a noncanonical motif of the p47phox C terminus. The electrostatic surfaces of both SH3 are very different, and therefore the binding preference for C-SH3p67 can be attributed to the polyproline motif recognition and particularly to the Arg-368p47 binding mode. The noncanonical motif contributes equally to interaction with both SH3. The influence of serine phosphorylation on residues 359/370 and 379 on the affinity for both SH3 domains has been checked. We conclude that contrarily to previous suggestions, phosphorylation of Ser-359/370 does not modify the SH3 binding affinity for both SH3, whereas phosphorylation of Ser-379 has a destabilizing effect on both interactions. Other mechanisms than a phosphorylation induced switch between the two SH3 must therefore take place for NADPH oxidase activation cascade to start.

The crystal structure of the minus-end-directed microtubule motor protein ncd reveals variable dimer conformations

BACKGROUND The kinesin superfamily of microtubule-associated motor proteins are important for intracellular transport and for cell division in eukaryotes. Conventional kinesins have the motor domain at the N terminus of the heavy chain and move towards the plus end of microtubules. The ncd protein is necessary for chromosome segregation in meiosis. It belongs to a subfamily of kinesins that have the motor domain at the C terminus and move towards the minus end of microtubules. RESULTS The crystal structure of dimeric ncd has been obtained at 2.9 A resolution from crystals with the C222(1) space group, with two independent dimers per asymmetric unit. The motor domains in these dimers are not related by crystallographic symmetry and the two ncd dimers have significantly different conformations. An alpha-helical coiled coil connects, and interacts with, the motor domains. CONCLUSIONS The ncd protein has a very compact structure, largely due to extended interactions of the coiled coil with the head domains. Despite this, we find that the overall conformation of the ncd dimer can be rotated by as much as 10 degrees away from that of the twofold-symmetric archetypal ncd. The crystal structures of conventional kinesin and of ncd suggest a structural rationale for the reversal of the direction of movement in chimeric kinesins.

Nature of S••••O interaction in short X−S••••O contacts: charge density experimental studies and theoretical interpretation

2-(2-Chlorobenzoylimino)-1,3-thiazolidine (I) and 3-benzoylimino-4-methyl-1,2,4-oxathiazane (II) present short intramolecular S ⋯· O contacts of 2.68 and 2.24 A, respectively. X-Ray and neutron diffraction at 122 K performed on (II), for comparison with (I), already studied, led to experimental charge density deformation maps which exhibit a strong peak around the sulphur atom. Using simple molecular orbital theory, it is possible to interpret the shortening of S ⋯· O distances in these compounds by σ-type interaction between the oxygen p and the sulphur p and d orbitals. The significant variation of the equilibrium S ⋯· O distance with the nature of the atom bonded to S is explained in terms of the strength of the coupling between X–S antibonding orbital and oxygen ione-pair orbitals.

Structure and RNA content of the prosomes

Duck erythroblast, prosomes were analysed by small angle neutron scattering (SANS), dynamic light scattering and (cryo‐)electron microscopy. A molecular weight of ≈ 720,000 ± 50,000, a radius of gyration of 64 ± 2 Å and a hydrodynamic radius of ≈ 86 Å were obtained. Electron micrographs show a hollow cylinder‐like particle with a diameter of 120 Å, n height of 170 Å and a diameter of 40 Å for the cavity, built of four discs, the two outer ones being more pronounced than those in the center. Results from SANS indicate less then 5% of RNA in the purified prosomes, but nuclease protection assays confirm its presence.

Structural studies of the glycine decarboxylase complex from pea leaf mitochondria.

The glycine decarboxylase complex consists of four different component enzymes (P-, H-, T- and L-proteins). The 14-kDa lipoamide-containing H-protein plays a pivotal role in the complete sequence of reactions since its prosthetic group (lipoic acid) interacts successively with the three other components of the complex and undergoes a cycle of reductive methylamination, methylamine transfer and electron transfer. The X-ray crystal structure of different forms of the H-protein has shown a unique conformation of the protein. This leads to the hypothesis of a three-dimensional recognition of the H-protein by the other components of the system and also by the ligase which lipoylates the H-protein. Striking structural similarities are observed between the H-protein and other lipoate domains of 2-oxo acid dehydrogenases and with the biotin carrier protein of acetyl-CoA carboxylase. In the H-protein, the lipoamide arm is free to move in the solvent when oxidized but is pivoted and tightly bound into a cleft at the protein surface when methylamine-loaded. This implies that the H-protein and the T-component form a stable complex during the catalytic transfer of the methylene unit to the tetrahydrofolate cofactor of the T-protein. This complex has been detected by small angle scattering experiments. In conclusion, in the glycine decarboxylase system, the lipoamide arm does not swing freely from one catalytic site to another as was proposed in other systems.

Crystallographic data for H-protein from the glycine decarboxylase complex

The H-protein is the pivotal enzyme of the glycine decarboxylase complex responsible for the oxidation of glycine by mitochondria. It has been extracted and purified from pea leaf mitochondria (Pisum sativum). Its molecular weight, based on the amino acid sequence, is 13.3 kDa and it crystallizes in the space group P3(1)21 (or its enantiomorph P3(2)21) with a = b = 57.14 (3) A, c = 137.11 (11) A. The crystals diffract until at least 3.5 A resolution.

Crystallographic data for the 9000 dalton wheat non-specific phospholipid transfer protein

The wheat non-specific phospholipid transfer protein belongs to a family of small proteins sharing a common pattern of four disulphide bridges. Its function in vivo is not known, but it has a high affinity to phospholipids and is involved in phospholipid transfer in vitro. The molecular weight is 9607, and it crystallizes in the space group P2(1) with a = 40.73 A, b = 112.11 A, c = 50.44 A and beta = 106.80 degrees. The crystals diffract to 3 A resolution.

Multiwavelength anomalous diffraction and diffuse scattering beam line project on a bending magnet at ESRF

The multiwavelength anomalous diffraction and diffuse scattering (D2AM) beam line project is designed for a 0.8‐T bending magnet at the ESRF for scattering experiments with multiwavelength anomalous techniques in the fields of (1) Materials Science (small angle and diffuse scattering) and (2) Crystallography of Biological Macromolecules (in particular MAD diffraction). Symmetric optics (a horizontally focusing double monochromator placed between two long, pseudoparabolic, vertically focusing mirrors) are planned. The optics, tested using the program shadow, should achieve a focus of 0.3×0.3 mm2 with an estimated flux of 1011 photons/s in a relative bandwidth of 2×10−4. Details of the beam line design are described in this note.

Crystallographic data for soybean hydrophobic protein

The soybean hydrophobic protein belongs to a family of proteins that contains a number of storage and phospholipid binding proteins. Its function is not known, but its overall hydrophobic nature is typical of many membrane proteins of similar size. The molecular weight is 8.3 x 10(3), and it crystallizes in the space group P2(1)2(1)2(1), with a = 52.01 A, b = 43.50 A and c = 28.80 A. The crystals diffract to 1.8 A resolution, and are thus suitable for X-ray structural studies.