RESUMEN
NKG2D, a homodimeric lectin-like receptor, is a unique stimulatory molecule that is found on natural killer cells,T cells and activated macrophages. The natural ligands for murine NKG2D are distant major histocompatibility complex homologs, retinoic acid early transcript (Rae1) and H-60 minor histocompatibility antigen. The crystal structure of the extracellular region of murine NKG2D reveals close homology with other C-type lectin receptors such as CD94, Ly49A, rat MBP-A and CD69. However, the precise mode of dimeric assembly varies among these natural killer receptors, as well as their surface topography and electrostatic properties. The NKG2D structure provides the first structural insights into the role and ligand specificity of this stimulatory receptor in the innate and adaptive immune system.
Asunto(s)
Cristalografía por Rayos X , Células Asesinas Naturales/inmunología , Receptores Inmunológicos/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dimerización , Disulfuros/química , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Lectinas/química , Lectinas Tipo C , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Subfamilia K de Receptores Similares a Lectina de Células NK , Unión Proteica , Isoformas de Proteínas/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Inmunológicos/inmunología , Receptores de Células Asesinas Naturales , Homología de Secuencia de AminoácidoRESUMEN
The oxygenase domain of the inducible nitric oxide synthase (iNOSox; residues 1-498) is a dimer that binds heme, L-arginine and tetrahydrobiopterin (H(4)B) and is the site for nitric oxide synthesis. We examined an N-terminal segment that contains a beta-hairpin hook, a zinc ligation center and part of the H(4)B-binding site for its role in dimerization, catalysis, and H(4)B and substrate interactions. Deletion mutagenesis identified the minimum catalytic core and indicated that an intact N-terminal beta-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, H(4)B 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 H(4)B. 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.
Asunto(s)
Biopterinas/análogos & derivados , Óxido Nítrico Sintasa/química , Óxido Nítrico Sintasa/metabolismo , Pterinas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Biopterinas/metabolismo , Bovinos , Dimerización , Drosophila , Humanos , Cinética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Óxido Nítrico Sintasa de Tipo II , Mutación Puntual , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , EspectrofotometríaRESUMEN
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.
Asunto(s)
Biopterinas/análogos & derivados , Óxido Nítrico Sintasa/química , Secuencia de Aminoácidos , Sitios de Unión , Biopterinas/metabolismo , Dimerización , Enlace de Hidrógeno , Datos de Secuencia Molecular , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Mutación Puntual , Relación Estructura-ActividadRESUMEN
Cytokine-inducible nitric oxide synthase (iNOS) is a homodimeric enzyme that generates nitric oxide (NO) and L-citrulline from L-arginine (L-Arg) and O2. The N-terminal oxygenase domain (amino acids 1-498; iNOSox) in each subunit binds heme, L-Arg, and tetrahydrobiopterin (H4B), is the site of NO synthesis, and is responsible for the dimeric interaction, which must occur to synthesize NO. In both cells and purified systems, iNOS dimer assembly is promoted by H4B, L-Arg, and L-Arg analogs. We examined the ability of imidazole and N-substituted imidazoles to promote or inhibit dimerization of heme-containing iNOSox monomers, or to affect iNOS dimerization in cells. Imidazole, 1-phenylimidazole, clotrimazole, and miconazole all bound to the iNOSox monomer heme iron. Imidazole and 1-phenylimidazole promoted iNOSox dimerization, whereas clotrimazole (30 microM) and miconazole (15 microM) did not, and instead inhibited dimerization normally promoted by L-Arg and H4B. Clotrimazole also bound to iNOSox dimers in the absence of L-Arg and H4B and caused their dissociation. When added to cells expressing iNOS, clotrimazole (50 microM) had no effect on iNOS protein expression but almost completely inhibited its dimerization and consequent NO synthesis over an 8-h culture period, without affecting calmodulin interaction with iNOS. Thus, imidazoles can promote or inhibit dimerization of iNOS both in vitro and in cells, depending on their structure. Bulky imidazoles like clotrimazole block NO synthesis by inhibiting assembly of the iNOS dimer, revealing a new means to control cellular NO synthesis.
Asunto(s)
Antifúngicos/farmacología , Clotrimazol/farmacología , Imidazoles/farmacología , Miconazol/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Animales , Línea Celular , Dimerización , Ratones , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo IIRESUMEN
Cytokine-inducible nitric-oxide (NO) synthase (iNOS) contains an oxygenase domain that binds heme, tetrahydrobiopterin, and L-arginine, and a reductase domain that binds FAD, FMN, calmodulin, and NADPH. Dimerization of two oxygenase domains allows electrons to transfer from the flavins to the heme irons, which enables O2 binding and NO synthesis from L-arginine. In an iNOS heterodimer comprised of one full-length subunit and an oxygenase domain partner, the single reductase domain transfers electrons to only one of two hemes (Siddhanta, U., Wu, C., Abu-Soud, H. M., Zhang, J., Ghosh, D. K., and Stuehr, D. J. (1996) J. Biol. Chem. 271, 7309-7312). Here, we characterize a pair of heterodimers that contain an L-Arg binding mutation (E371A) in either the full-length or oxygenase domain subunit to identify which heme iron becomes reduced. The E371A mutation prevented L-Arg binding to one oxygenase domain in each heterodimer but did not affect the L-Arg affinity of its oxygenase domain partner and did not prevent heme iron reduction in any case. The mutation prevented NO synthesis when it was located in the oxygenase domain of the adjacent subunit but had no effect when in the oxygenase domain in the same subunit as the reductase domain. Resonance Raman characterization of the heme-L-Arg interaction confirmed that E371A only prevents L-Arg binding in the mutated oxygenase domain. Thus, flavin-to-heme electron transfer proceeds exclusively between adjacent subunits in the heterodimer. This implies that domain swapping occurs in an iNOS dimer to properly align reductase and oxygenase domains for NO synthesis.
Asunto(s)
Flavinas/metabolismo , Hemo/metabolismo , Óxido Nítrico Sintasa/metabolismo , Arginina/metabolismo , Sitios de Unión , Catálisis , Dimerización , Transporte de Electrón , Escherichia coli , NADP/metabolismo , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa de Tipo II , Oxidación-Reducción , Mutación Puntual , Conformación Proteica , Espectrofotometría Atómica , Espectrometría RamanRESUMEN
Physical and sexual abuse are increasingly recognized as common harmful experiences for women. We surveyed 828 women veterans seeking care at the Baltimore Veterans Affairs Medical Center to determine the prevalence of physical and sexual abuse experiences, both during and outside of military service. Data were collected through an anonymous, mailed questionnaire, with a response rate of 52%. Sixty-eight percent of respondents reported at least one form of abuse, and 27% reported all three. Sexual abuse was most common (55%), followed by physical abuse (48%), and rape (41%). Enlisted women, women younger than 50, and single, separated, or divorced women were significantly more likely to report abusive experiences. Over 40% of the women reporting abuse were never victimized while on active duty, and these women were less likely to receive counseling. Physical and sexual abuse experiences are disturbingly common among women veterans and demonstrate the need for additional services to assist these women.
PIP: A survey to determine the prevalence of physical and sexual abuse experiences, during and outside of military service, was conducted among 828 women veterans at the Baltimore Veterans Affairs Medical Center. Data collection was through anonymous, mailed questionnaire. Three questions were used to elicit histories of physical abuse, sexual abuse, and rape. From the survey, 429 completed forms (52%) were returned. Most of the veterans had at least some college education and about 50% served 4 or more years on active duty. About 68% of the respondents reported at least one form of victimization, while 27% reported to have undergone all three forms, of which sexual abuse was the most common, followed by physical abuse and then rape. It was in adulthood that all three forms of abuse took place with one-third of the women reporting victimization during active duty. However, 50% of the women veterans reporting physical abuse, 44% reporting sexual abuse, and 52% reporting rape were never victimized during their military careers. Single women and divorced women were more likely to report victimization. In conclusion, physical and sexually abused veteran women were the ones more likely seeking care at the center.