Brian R. Crane

Structure of CheA, a Signal-Transducing Histidine Kinase

Histidine kinases allow bacteria, plants, and fungi to sense and respond to their environment. The 2.6 A resolution crystal structure of Thermotoga maritima CheA (290-671) histidine kinase reveals a dimer where the functions of dimerization, ATP binding, and regulation are segregated into domains. The kinase domain is unlike Ser/Thr/Tyr kinases but resembles two ATPases, Gyrase B and Hsp90. Structural analogies within this superfamily suggest that the P1 domain of CheA provides the nucleophilic histidine and activating glutamate for phosphotransfer. The regulatory domain, which binds the homologous receptor-coupling protein CheW, topologically resembles two SH3 domains and provides different protein recognition surfaces at each end. The dimerization domain forms a central four-helix bundle about which the kinase and regulatory domains pivot on conserved hinges to modulate transphosphorylation. Different subunit conformations suggest that relative domain motions link receptor response to kinase activity.

Sulfite reductase structure at 1.6 A: evolution and catalysis for reduction of inorganic anions.

Fundamental chemical transformations for biogeochemical cycling of sulfur and nitrogen are catalyzed by sulfite and nitrite reductases. The crystallographic structure of Escherichia coli sulfite reductase hemoprotein (SiRHP), which catalyzes the concerted six-electron reductions of sulfite to sulfide and nitrite to ammonia, was solved with multiwavelength anomalous diffraction (MAD) of the native siroheme and Fe 4S4 cluster cofactors, multiple isomorphous replacement, and selenomethionine sequence markers. Twofold symmetry within the 64-kilodalton polypeptide generates a distinctive three-domain α/β fold that controls cofactor assembly and reactivity. Homology regions conserved between the symmetry-related halves of SiRHP and among other sulfite and nitrite reductases revealed key residues for stability and function, and identified a sulfite or nitrite reductase repeat (SNiRR) common to a redox-enzyme superfamily. The saddle-shaped siroheme shares a cysteine thiolate ligand with the Fe4S4 cluster and ligates an unexpected phosphate anion. In the substrate complex, sulfite displaces phosphate and binds to siroheme iron through sulfur. An extensive hydrogen-bonding network of positive side chains, water molecules, and siroheme carboxylates activates S-O bonds for reductive cleavage.

Nucleotide binding by the histidine kinase CheA

To probe the structural basis for protein histidine kinase (PHK) catalytic activity and the prospects for PHK-specific inhibitor design, we report the crystal structures for the nucleotide binding domain of Thermotoga maritima CheA with ADP and three ATP analogs (ADPNP, ADPCP and TNP-ATP) bound with either Mg2+ or Mn2+. The conformation of ADPNP bound to CheA and related ATPases differs from that reported in the ADPNP complex of PHK EnvZ. Interactions of the active site with the nucleotide γ-phosphate and its associated Mg2+ ion are linked to conformational changes in an ATP-lid that could mediate recognition of the substrate domain. The inhibitor TNP-ATP binds CheA with its phosphates in a nonproductive conformation and its adenine and trinitrophenyl groups in two adjacent binding pockets. The trinitrophenyl interaction may be exploited for designing CheA-targeted drugs that would not interfere with host ATPases.

Cloning, expression, and characterization of a nitric oxide synthase protein from Deinococcus radiodurans

We cloned, expressed, and characterized a hemeprotein from Deinococcus radiodurans (D. radiodurans NO synthase, deiNOS) whose sequence is 34% identical to the oxygenase domain of mammalian NO synthases (NOSoxys). deiNOS was dimeric, bound substrate Arg and cofactor tetrahydrobiopterin, and had a normal heme environment, despite its missing N-terminal structures that in NOSoxy bind Zn2+ and tetrahydrobiopterin and help form an active dimer. The deiNOS heme accepted electrons from a mammalian NOS reductase and generated NO at rates that met or exceeded NOSoxy. Activity required bound tetrahydrobiopterin or tetrahydrofolate and was linked to formation and disappearance of a typical heme-dioxy catalytic intermediate. Thus, bacterial NOS-like proteins are surprisingly similar to mammalian NOSs and broaden our perspective of NO biochemistry and function.

The relationship between structure and function for the sulfite reductases

The six-electron reductions of sulfite to sulfide and nitrite to ammonia, fundamental to early and contemporary life, are catalyzed by diverse sulfite and nitrite reductases that share an unusual prosthetic assembly in their active centers, namely siroheme covalently linked to an Fe4S4 cluster. The recently determined crystallographic structure of the sulfite reductase hemoprotein from Escherichia coli complements extensive biochemical and spectroscopic studies in revealing structural features that are key for the catalytic mechanisms and in suggesting a common symmetric structural unit for this diverse family of enzymes.

N-Terminal Domain Swapping and Metal Ion Binding in Nitric Oxide Synthase Dimerization

Nitric oxide synthase oxygenase domains (NOSox) must bind tetrahydrobiopterin and dimerize to be active. New crystallographic structures of inducible NOSox reveal that conformational changes in a switch region (residues 103–111) preceding a pterin‐binding segment exchange N‐terminal β‐hairpin hooks between subunits of the dimer. N‐terminal hooks interact primarily with their own subunits in the ‘unswapped’ structure, and two switch region cysteines (104 and 109) from each subunit ligate a single zinc ion at the dimer interface. N‐terminal hooks rearrange from intra‐ to intersubunit interactions in the ‘swapped structure’, and Cys109 forms a self‐symmetric disulfide bond across the dimer interface. Subunit association and activity are adversely affected by mutations in the N‐terminal hook that disrupt interactions across the dimer interface only in the swapped structure. Residue conservation and electrostatic potential at the NOSox molecular surface suggest likely interfaces outside the switch region for electron transfer from the NOS reductase domain. The correlation between three‐dimensional domain swapping of the N‐terminal hook and metal ion release with disulfide formation may impact inducible nitric oxide synthase (i)NOS stability and regulation in vivo.

Inducible nitric oxide synthase: role of the N‐terminal β‐hairpin hook and pterin‐binding segment in dimerization and tetrahydrobiopterin interaction

The oxygenase domain of the inducible nitric oxide synthase (iNOSox; residues 1–498) is a dimer that binds heme, L‐arginine and tetrahydrobiopterin (H4B) and is the site for nitric oxide synthesis. We examined an N‐terminal segment that contains a β‐hairpin hook, a zinc ligation center and part of the H4B‐binding site for its role in dimerization, catalysis, and H4B and substrate interactions. Deletion mutagenesis identified the minimum catalytic core and indicated that an intact N‐terminal β‐hairpin hook is essential. Alanine screening mutagenesis of conserved residues in the hook revealed five positions (K82, N83, D92, T93 and H95) where native properties were perturbed. Mutants fell into two classes: (i) incorrigible mutants that disrupt side‐chain hydrogen bonds and packing interactions with the iNOSox C‐terminus (N83, D92 and H95) and cause permanent defects in homodimer formation, H4B binding and activity; and (ii) reformable mutants that destabilize interactions of the residue main chain (K82 and T93) with the C‐terminus and cause similar defects that were reversible with high concentrations of H4B. Heterodimers comprised of a hook‐defective iNOSox mutant subunit and a full‐length iNOS subunit were active in almost all cases. This suggests a mechanism whereby N‐terminal hooks exchange between subunits in solution to stabilize the dimer.

Tryptophan 409 controls the activity of neuronal nitric-oxide synthase by regulating nitric oxide feedback inhibition.

The heme of neuronal nitric-oxide synthase participates in oxygen activation but also binds self-generated NO during catalysis resulting in reversible feedback inhibition. We utilized point mutagenesis to investigate if a conserved tryptophan residue (Trp-409), which engages in π-stacking with the heme and hydrogen bonds to its axial cysteine ligand, helps control catalysis and regulation by NO. Surprisingly, mutants W409F and W409Y were hyperactive compared with the wild type regarding NO synthesis without affecting cytochrome c reduction, reductase-independentN-hydroxyarginine oxidation, or Arg and tetrahydrobiopterin binding. In the absence of Arg, NADPH oxidation measurements showed that electron flux through the heme was actually slower in the Trp-409 mutants than in wild-type nNOS. However, little or no NO complex accumulated during NO synthesis by the mutants, as opposed to the wild type. This difference was potentially related to mutants forming unstable 6-coordinate ferrous-NO complexes under anaerobic conditions even in the presence of Arg and tetrahydrobiopterin. Thus, Trp-409 mutations minimize NO feedback inhibition by preventing buildup of an inactive ferrous-NO complex during the steady state. This overcomes the negative effect of the mutation on electron flux and results in hyperactivity. Conservation of Trp-409 among different NOS suggests that the ability of this residue to regulate heme reduction and NO complex formation is important for enzyme physiologic function.

Mutational Analysis of the Tetrahydrobiopterin-binding Site in Inducible Nitric-oxide Synthase

Inducible nitric-oxide synthase (iNOS) is a hemeprotein that requires tetrahydrobiopterin (H4B) for activity. The influence of H4B on iNOS structure-function is complex, and its exact role in nitric oxide (NO) synthesis is unknown. Crystal structures of the mouse iNOS oxygenase domain (iNOSox) revealed a unique H4B-binding site with a high degree of aromatic character located in the dimer interface and near the heme. Four conserved residues (Arg-375, Trp-455, Trp-457, and Phe-470) engage in hydrogen bonding or aromatic stacking interactions with the H4B ring. We utilized point mutagenesis to investigate how each residue modulates H4B function. All mutants contained heme ligated to Cys-194 indicating no deleterious effect on general protein structure. Ala mutants were monomers except for W457A and did not form a homodimer with excess H4B and Arg. However, they did form heterodimers when paired with a full-length iNOS subunit, and these were either fully or partially active regarding NO synthesis, indicating that preserving residue identities or aromatic character is not essential for H4B binding or activity. Aromatic substitution at Trp-455 or Trp-457 generated monomers that could dimerize with H4B and Arg. These mutants bound Arg and H4B with near normal affinity, but Arg could not displace heme-bound imidazole, and they had NO synthesis activities lower than wild-type in both homodimeric and heterodimeric settings. Aromatic substitution at Phe-470 had no significant effects. Together, our work shows how hydrogen bonding and aromatic stacking interactions of Arg-375, Trp-457, Trp-455, and Phe-470 influence iNOSox dimeric structure, heme environment, and NO synthesis and thus help modulate the multiple effects of H4B.

Sensitizer-linked substrates and ligands: ruthenium probes of cytochrome P450 structure and mechanism.

Publisher Summary This chapter describes methodologies for employing sensitizer-linked substrates and ligands as probes of P450 structure and mechanism. Sensitizer-linked substrates target cytochromes P450 in a background of other heme enzymes, and their demonstrated biosensing and substrate screening capabilities may facilitate future drug design efforts. Unlike many structural probes of the P450 hydrophobic pocket, sensitizer-linked substrates bind reversibly and may undergo modest turnover during biological catalysis. Resonance Raman spectroscopy interrogates the heme environment in P450 Fe 2+ -CO Ru-substrate complexes. Ru-substrates complement existing mechanistic probes by providing an efficient ET pathway to the P450 heme through the hydrocarbon linker. Because of rate-limiting ET in the natural enzymatic system reactive Fe-peroxy and ferryl intermediates in P450 catalytic cycles are not readily observable. A general procedure for generating sensitizer-linked substrates is discussed in the chapter through a figure. This protocol may be generalized to include any combination of photosensitizer, linker, and substrate/ligand that targets the desired P450 active site. Ones have synthesized numerous compounds: Ru and Os photosensitizers with substituted bipyridine, terpyridine, phenanthroline, and imidazole ligands; alkyl, polyethylene glycol, perfluorobiphenyl, and polyxylyl linkers; attached via amide, ether, or amino bonds to substrates (adamantane, ethyl benzene, borneol, norbornane, thioanisole styrene) or imidazole ligands.