Structural insight into the evolution of a new chemokine family from zebrafish.

The mammalian chemokine family is segregated into four families - CC, CXC, CX3C, and XC-based on the arrangement of cysteines and the corresponding disulfides. Sequencing of the Danio rerio (zebrafish) genome has identified more than double the amount of human chemokines with the absence of the CX3C family and the presence of a new family, CX. The only other family with a single cysteine in the N-terminal region is the XC family. Human lymphotactin (XCL1) has two interconverting structures due to dynamic changes that occur in the protein. Similar to an experiment with XCL1 that identified the two structural forms, we probed for multiple forms of zCXL1 using heparin affinity. The results suggest only a single form of CXL1 is present. We used sulfur-SAD phasing to determine the three-dimensional structure CXL1. Zebrafish CXL1 (zCXL1) has three disulfides that appear to be important for a stable structure. One disulfide is common to all chemokines except those that belong to the XC family, another is similar to a subset of CC chemokines containing three disulfides, but the third disulfide is unique to the CX family. We analyzed the electrostatic potential of the zCXL1 structure and identified the likely heparin-binding site for glycosaminoglycans (GAGs). zCXL1 has a similar sequence identity with human CCL5 and CXCL12, but the structure is more related to CCL5. Our structural analysis supports the phylogenetic and genomic studies on the evolution of the CXL family.

Structural basis for cyclic-nucleotide selectivity and cGMP-selective activation of PKG I.

Cyclic guanosine monophosphate (cGMP) and cyclic AMP (cAMP)-dependent protein kinases (PKG and PKA) are closely related homologs, and the cyclic nucleotide specificity of each kinase is crucial for keeping the two signaling pathways segregated, but the molecular mechanism of cyclic nucleotide selectivity is unknown. Here, we report that the PKG Iß C-terminal cyclic nucleotide binding domain (CNB-B) is highly selective for cGMP binding, and we have solved crystal structures of CNB-B with and without bound cGMP. These structures, combined with a comprehensive mutagenic analysis, allowed us to identify Leu296 and Arg297 as key residues that mediate cGMP selectivity. In addition, by comparing the cGMP bound and unbound structures, we observed large conformational changes in the C-terminal helices in response to cGMP binding, which were stabilized by recruitment of Tyr351 as a "capping residue" for cGMP. The observed rearrangements of the C-terminal helices provide a mechanical insight into release of the catalytic domain and kinase activation.

Away from the edge II: in-house Se-SAS phasing with chromium radiation.

Recently, the demands of high-throughput macromolecular crystallography have driven continuous improvements in phasing methods, data-collection protocols and many other technologies. Single-wavelength anomalous scattering (SAS) phasing with chromium X-ray radiation opens a new possibility for phasing a protein with data collected in-house and has led to several successful examples of de novo structure solution using only weak anomalous scatterers such as sulfur. To further reduce data-collection time and make SAS phasing more robust, it is natural to combine selenomethionine-derivatized protein (SeMet protein) with Cr Kalpha radiation to take advantage of the larger anomalous scattering signal from selenium (f'' = 2.28 e(-)) compared with sulfur (f'' = 1.14 e(-)). As reported herein, the crystal structure of a putative chorismate mutase from Clostridium thermocellum was determined using Se-SAS with Cr Kalpha radiation. Each protein molecule contains eight selenomethionine residues in 148 amino-acid residues, providing a calculated Bijvoet ratio of about 3.5% at the Cr Kalpha wavelength. A single data set to 2.2 A resolution with approximately ninefold redundancy was collected using an imaging-plate detector coupled with a Cr source. Structure solution, refinement and deposition to the Protein Data Bank were performed within 9 h of the availability of the scaled diffraction data. The procedure used here is applicable to many other proteins and promises to become a routine pathway for in-house high-throughput crystallography.

Structural basis for herbicidal inhibitor selectivity revealed by comparison of crystal structures of plant and mammalian 4-hydroxyphenylpyruvate dioxygenases.

A high degree of selectivity toward the target site of the pest organism is a desirable attribute for new safer agrochemicals. To assist in the design of novel herbicides, we determined the crystal structures of the herbicidal target enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD; EC 1.13.11.27) from the plant Arabidopsis thaliana with and without an herbicidal benzoylpyrazole inhibitor that potently inhibits both plant and mammalian HPPDs. We also determined the structure of a mammalian (rat) HPPD in complex with the same nonselective inhibitor. From a screening campaign of over 1000 HPPD inhibitors, six highly plant-selective inhibitors were found. One of these had remarkable (>1600-fold) selectivity toward the plant enzyme and was cocrystallized with Arabidopsis HPPD. Detailed comparisons of the plant and mammalian HPPD-ligand structures suggest a structural basis for the high degree of plant selectivity of certain HPPD inhibitors and point to design strategies to obtain potent and selective inhibitors of plant HPPD as agrochemical leads.

Crystal structure of the oxygen-dependant coproporphyrinogen oxidase (Hem13p) of Saccharomyces cerevisiae.

Coproporphyrinogen oxidase (CPO) is an essential enzyme that catalyzes the sixth step of the heme biosynthetic pathway. Unusually for heme biosynthetic enzymes, CPO exists in two evolutionarily and mechanistically distinct families, with eukaryotes and some prokaryotes employing members of the highly conserved oxygen-dependent CPO family. Here, we report the crystal structure of the oxygen-dependent CPO from Saccharomyces cerevisiae (Hem13p), which was determined by optimized sulfur anomalous scattering and refined to a resolution of 2.0 A. The protein adopts a novel structure that is quite different from predicted models and features a central flat seven-stranded anti-parallel sheet that is flanked by helices. The dimeric assembly, which is seen in different crystal forms, is formed by packing of helices and a short isolated strand that forms a beta-ladder with its counterpart in the partner subunit. The deep active-site cleft is lined by conserved residues and has been captured in open and closed conformations in two different crystal forms. A substratesized cavity is completely buried in the closed conformation by the approximately 8-A movement of a helix that forms a lid over the active site. The structure therefore suggests residues that likely play critical roles in catalysis and explains the deleterious effect of many of the mutations associated with the disease hereditary coproporphyria.