Refined crystal structure of ferrocytochrome c2 from Rhodopseudomonas viridis at 1.6 A resolution.

The three-dimensional structure of ferrocytochrome c2 from the purple photosynthetic bacterium Rhodopseudomonas viridis has been refined to a final R-factor of 18.2% for 15,014 unique reflections collected by synchrotron radiation between 6.0 and 1.6 A resolution. The refined model includes 107 amino acid residues, one heme prosthetic group and 125 water molecules. The root-mean-square deviations from the ideal bond lengths and angles were 0.014 A and 3.0 degrees, respectively. The atomic coordinate error was estimated to be less than 0.3 A. A structure comparison of this cytochrome c2 with those of the other c-type cytochromes demonstrated that these cytochromes exhibit a high degree of structural similarity with the exception of the surface loop and the terminal region of the polypeptide chain. The deletion of an intrahelical amino residue distorted the conformation of the alpha-helix and it divided into two pieces. The C-terminal extension of the polypeptide chain caused significant conformational changes of the contact residues compared with the other c-type cytochromes. Of the water molecules conserved in various c-type cytochromes, two are located internally in the vicinity of the heme group. One of these water molecules found in this cytochrome c2 is evolutionarily conserved among eukaryotic cytochromes c. This water molecule is located in the heme proximate environment in a position similar to that of eukaryotic cytochromes c. The position of this water molecule is associated with the oxidation state of the heme iron in electron transfer.

Neutralizing epitopes on the extracellular interferon gamma receptor (IFNgammaR) alpha-chain characterized by homolog scanning mutagenesis and X-ray crystal structure of the A6 fab-IFNgammaR1-108 complex.

The extracellular interferon gamma receptor alpha-chain comprises two immunoglobulin-like domains, each with fibronectin type-III topology, which are responsible for binding interferon gamma at the cell surface. The epitopes on the human receptor recognized by three neutralizing antibodies, A6, gammaR38 and gammaR99, have been mapped by homolog scanning mutagenesis. In this way, a loop connecting beta-strands C and C' in the N-terminal domain was identified as a key component of the epitopes bound by A6 and gammaR38, whereas gammaR99 binds to the C-terminal domain in a region including strands A and B and part of the large C'E loop. The epitope for A6 was confirmed in a crystal structure of a complex between a recombinant N-terminal receptor domain and the Fab fragment from A6, determined by X-ray diffraction to 2.8 A resolution. The antibody-antigen interface buries 1662 A2 of protein surface, including 22 antibody residues from five complementarity determining regions, primarily through interactions with the CC' surface loop of the receptor. The floor of the antigen binding cavity is formed mainly by residues from CDR L3 and CDR H3 while a surrounding ridge is formed by residues from all other CDRs except L2. Many potential polar interactions, as well as 13 aromatic side-chains, four in VL, six in VH and three in the receptor, are situated at the interface. The surface of the receptor contacted by A6 overlaps to a large extent with that contacted by interferon-gamma, in the ligand-receptor complex. However, the conformation of this epitope is very different in the two complexes, demonstrating that conformational mobility in a surface loop on this cytokine receptor permits steric and electrostatic complementarity to two quite differently shaped binding sites.

Radiation-induced meningiomas after BNCT in patients with malignant glioma.

Of the 180 patients with malignant brain tumors whom we treated with boron neutron capture therapy (BNCT) since 1968, only one (0.56%) developed multiple radiation-induced meningiomas. The parasagittal meningioma that had received 42 Gy (w) for BNCT showed more rapid growth on Gd-enhanced MRI scans and more atypical features on histopathologic studies than the temporal convexity tumor that had received 20 Gy (w). Long-term follow up MRI studies are necessary in long-survivors of malignant brain tumors treated by BNCT.

Crystal structure of the oxidized cytochrome c(2) from Blastochloris viridis.

The crystal structure of the oxidized cytochrome c(2) from Blastochloris (formerly Rhodopseudomonas) viridis was determined at 1.9 A resolution. Structural comparison with the reduced form revealed significant structural changes according to the oxidation state of the heme iron. Slight perturbation of the polypeptide chain backbone was observed, and the secondary structure and the hydrogen patterns between main-chain atoms were retained. The oxidation state-dependent conformational shifts were localized in the vicinity of the methionine ligand side and the propionate group of the heme. The conserved segment of the polypeptide chain in cytochrome c and cytochrome c(2) exhibited some degree of mobility, interacting with the heme iron atom by the hydrogen bond network. These results indicate that the movement of the internal water molecule conserved in various c-type cytochromes drives the adjustments of side-chain atoms of nearby residue, and the segmental temperature factor changes along the polypeptide chain.

Structural similarity of cytochrome c2 from Rhodopseudomonas viridis to mitochondrial cytochromes c revealed by its crystal structure at 2.7 A resolution.

The crystal structure of cytochrome c2 from Rhodopseudomonas viridis has been refined using molecular dynamics and restrained least-squares methods to a crystallographic R-factor of 0.216 at 2.7 A resolution. A structural comparison between Rps. viridis cytochrome c2 and the other bacterial cytochromes c2 or mitochondrial cytochromes c indicates that the overall protein foldings are very similar to each other with the exception of the surface loop and terminal region of the polypeptide chain. However, the position and hydrogen-bond pattern of the evolutionarily conserved water molecule buried within the heme binding pocket in Rps. viridis cytochrome c2 are common to those in the mitochondrial cytochromes c. This fact indicates that Rps. viridis cytochrome c2 is structurally more similar to mitochondrial cytochromes c than to the other bacterial cytochromes c2.

Structure-based design of beta-lactamase inhibitors. 1. Synthesis and evaluation of bridged monobactams.

Bridged monobactams are novel, potent, mechanism-based inhibitors of class C beta-lactamases, designed using X-ray crystal structures of the enzymes. They stabilize the acyl-enzyme intermediate by blocking access of water to the enzyme-inhibitor ester bond. Bridged monobactams are selective class C beta-lactamase inhibitors, with half-inhibition constants as low as 10 nM, and are less effective against class A and class B enzymes (half-inhibition constants > 100 microM) because of the different hydrolysis mechanisms in these classes of beta-lactamases. The stability of the acyl-enzyme complexes formed with class C beta-lactamases (half-lives up to 2 days were observed) enabled determination of their crystal structures. The conformation of the inhibitor moiety was close to that predicted by molecular modeling, confirming a simple reaction mechanism, unlike those of known beta-lactamase inhibitors such as clavulanic acid and penam sulfones, which involve secondary rearrangements. Synergy between the bridged monobactams and beta-lactamase-labile antibiotics could be observed when such combinations were tested against strains of Enterobacteriaceae that produce large amounts of class C beta-lactamases. The minimal inhibitory concentration of the antibiotic of more than 64 mg/L could be decreased to 0.25 mg/L in a 1:4 combination with the inhibitor.

Comparison of the binding sites for high-potential iron-sulfur protein and cytochrome c on the tetraheme cytochrome subunit bound to the bacterial photosynthetic reaction center.

A tetraheme cytochrome subunit bound to the photosynthetic reaction center (RC) of purple bacterium, Rubrivivax gelatinosus, interacts with two types of soluble electron donors, cytochromes c and high-potential iron-sulfur protein (HiPIP), at a binding domain in the vicinity of low-potential heme 1, the fourth heme from the special pair of bacteriochlorophyll. To clarify the mechanism of the interaction, the domain around heme 1 was examined using site-directed mutants that changed the surface charge in the region within 20 A from the heme edge. In the case of the interaction with soluble cytochrome c, a strong dependence on the sign of the introduced charge was observed in all mutants: positive charge inhibited the reaction rate, whereas additional negative charge accelerated it. This confirmed the electrostatic nature of the binding. Interaction with HiPIP was inhibited by a limited number of mutations at the close vicinity of heme 1, and no acceleration was observed (the effects of some mutations were independent of the sign of the introduced charge). The acidic residues which were critically important for the binding of cytochrome c showed much less contribution to the binding of HiPIP. The binding site for HiPIP appears to be mostly formed by uncharged and hydrophobic residues, occupying a significantly smaller area than the cytochrome-c-binding site. It is proposed that the docking of HiPIP to the RC in Rvi. gelatinosus is primarily controlled by hydrophobic contacts between protein surfaces, thus differing from the electrostatic mode of the RC-cytochrome c interaction.

Solid-phase synthesis of caged peptides using tyrosine modified with a photocleavable protecting group: application to the synthesis of caged neuropeptide Y.

A simple method for the synthesis of caged peptides using a novel derivative of tyrosine, N-Fmoc-O-(2-nitrobenzyl)-tyrosine, is described. The derivative of tyrosine can be incorporated at any position in an amino acid sequence by solid-phase peptide synthesis under the condition for Fmoc chemistry, and caged peptides that contain nitrobenzyl group on the side chain of tyrosine residue can be obtained. The nitrobenzyl group can be photocleaved by UV irradiation and the half life of the intermediate during photolysis is approximately 7 microseconds. The method was successfully applied to the synthesis of caged neuropeptide Y (NPY). The binding affinity of the caged NPY for the Y1 receptor was one or two orders of magnitude lower than that of intact NPY, but it increased to the value for intact NPY upon irradiation by UV light.

Different mechanisms of the binding of soluble electron donors to the photosynthetic reaction center of Rubrivivax gelatinosus and Blastochloris viridis.

The tetraheme cytochrome subunits of the photosynthetic reaction centers (RCs) in two species of purple bacteria, Rubrivivax gelatinosus and Blastochloris (Rhodopseudomonas) viridis, were compared in terms of their capabilities to bind different electron-donor proteins. The wild-type RCs from both species and mutated forms of R. gelatinosus RCs (with amino acid substitutions introduced to the binding domain for electron-donor proteins) were tested for their reactivity with soluble cytochromes and high potential iron-sulfur protein. Cytochromes from both species were good electron donors to the B. viridis RC and the R. gelatinosus RC. The reactivity in the R. gelatinosus RC showed a clear dependence on the polarity of the charges introduced to the binding domain, indicating the importance of the electrostatic interactions. In contrast, high potential iron-sulfur protein, presumed to operate according to the hydrophobic mechanism of binding, reacted significantly only with the R. gelatinosus RC. Evolutionary substitution of amino acids in a region of the binding domain on the cytochrome subunit surface probably caused the change in the principal mode of protein-protein interactions in the electron-transfer chains.

Interaction site for soluble cytochromes on the tetraheme cytochrome subunit bound to the bacterial photosynthetic reaction center mapped by site-directed mutagenesis.

The crystallographic structure of the Blastochloris (formerly called Rhodopseudomonas) viridis tetraheme cytochrome subunit bound to the photosynthetic reaction center (RC) suggests that all four hemes are located close enough to the surface of the protein to accept electrons from soluble cytochrome c2. To identify experimentally the site of this reaction we prepared site-directed mutants of Rubrivivax gelatinosus RCs with surface charge substitutions in the bound cytochrome subunit and studied the kinetics of their reduction by soluble cytochromes (mitochondrial horse cytochrome c, Blc. viridis cytochrome c2, and Rvi. gelatinosus cytochrome c8). In comparison with the wild-type, the mutants E79K (glutamate-79 substituted by lysine), E93K (glutamate-93 substituted by lysine), and E85K (glutamate-85 substituted by lysine) located near the solvent-exposed edge of low-potential heme 1, the fourth heme from the special pair of bacteriochlorophyll, exhibited decreased second-order rate constants for the reaction between the tetraheme subunit and the soluble cytochromes. Double charge substitutions in this region: E79K/E85K (glutamate-79 and -85 both replaced by lysine) and E93K/E85K (glutamate-93 and -85 both replaced by lysine) appeared to show an additive inhibitory effect. Mutations in other charged regions did not alter the kinetics of electron transfer between bound and soluble cytochromes. In light of the available structural information on Blc. viridis RC, these results indicate that the cluster of acidic residues immediately surrounding the distal heme 1 of the RC-bound tetraheme subunit forms an electrostatically favorable binding site for soluble cytochromes. Thus, all four hemes in the subunit seem to be directly involved in the electron transfer toward the photo-oxidized special pair of bacteriochlorophyll. On the basis of these findings, a model is proposed for the hypothetical cytochrome c2-RC transient complex for Blc. viridis.

Application of an automatic molecular-replacement procedure to crystal structure analysis of cytochrome c2 from Rhodopseudomonas viridis.

An automatic molecular-replacement procedure has been applied to solve the crystal structure of cytochrome c(2) from Rhodopseudomonas viridis. The structure was solved on the basis of the structure of tuna cytochrome c as a search model using an automatic processing program system, AUTOMR. The refinements by molecular dynamics and restrained least-squares methods result in a current crystallographic R factor of 0.219 for diffraction data at 3 A resolution.

Design and synthesis of novel benzofurans as a new class of antifungal agents targeting fungal N-myristoyltransferase. Part 1.

Potent and selective Candida albicans N-myristoyltransferase (CaNmt) inhibitors have been identified through optimization of a lead compound 1 discovered by random screening. The inhibitor design is based on the crystal structure of the CaNmt complex with compound (S)-3 and structure-activity relationships (SARs) have been clarified. Modification of the C-4 side chain of 1 has led to the discovery of a potent and selective CaNmt inhibitor 11 (RO-09-4609), which exhibits antifungal activity against C. albicans in vitro.