RESUMEN
Dengue virus (DENV), a member of the family Flaviviridae, presents a tremendous threat to global health since an estimated 2.5 billion people worldwide are at risk for epidemic transmission. DENV infections are primarily restricted to sub-tropical and tropical regions; however, there is concern that the virus will spread into new regions including the United States. There are no approved antiviral drugs or vaccines to combat dengue infection, although DENV vaccines have entered Phase 3 clinical trials. Drug discovery and development efforts against DENV and other viral pathogens must overcome specificity, efficacy, safety, and resistance challenges before the shortage of licensed drugs to treat viral infections can be relieved. Current drug discovery methods are largely inefficient and thus relatively ineffective at tackling the growing threat to public health presented by emerging and remerging viral pathogens. This review discusses current and newly implemented structure-based computational efforts to discover antivirals that target the DENV NS3 protease, although it is clear that these computational tools can be applied to most disease targets.
Asunto(s)
Virus del Dengue/enzimología , Dengue/tratamiento farmacológico , Descubrimiento de Drogas , Inhibidores de Proteasas/uso terapéutico , Serina Endopeptidasas/efectos de los fármacos , Animales , Ensayos Clínicos Fase III como Asunto , Biología Computacional/métodos , Vacunas contra el Dengue/uso terapéutico , Virus del Dengue/efectos de los fármacos , Virus del Dengue/inmunología , Relación Estructura-ActividadRESUMEN
Although alphaviruses have been extensively studied as model systems for the structural organization of enveloped viruses, no structures exist for the phylogenetically distinct eastern equine encephalomyelitis (EEE)-Venezuelan equine encephalomyelitis (VEE) lineage of New World alphaviruses. Here we report the 25-A structure of VEE virus, obtained from electron cryomicroscopy and image reconstruction. The envelope spike glycoproteins of VEE virus have a T=4 icosahedral arrangement, similar to that observed in Old World Sindbis, Semliki Forest, and Ross River alphaviruses. However, VEE virus has pronounced differences in its nucleocapsid structure relative to nucleocapsid structures repeatedly observed in Old World alphaviruses.
Asunto(s)
Virus de la Encefalitis Equina Venezolana/ultraestructura , Alphavirus/ultraestructura , Animales , Evolución BiológicaRESUMEN
Mammalian pyruvate kinase (PK) is a four-domain enzyme that is active as a homo-tetramer. Tissue-specific isozymes of PK exhibit distinct levels of allosteric regulation. PK expressed in muscle tissue (M1-PK) shows hyperbolic steady-state kinetics, whereas PK expressed in kidney tissue (M2-PK) displays sigmoidal kinetics. Rabbit M1 and M2-PK are isozymes whose sequences differ in only 22 out of 530 residues per subunit, and these changes are localized in an inter-subunit interface. Previous studies have shown that a single amino acid mutation to M1-PK at either the Y (S402P) or Z (T340 M) subunit interface can confer a level of allosteric regulation that is intermediate to M1-PK and M2-PK. In an effort to elucidate the roles of the inter-subunit interaction in signal transmission and the functional/structural connectivity between these interfaces, the S402P mutant of M1-PK was crystallized and its structure resolved to 2.8 A. Although the overall S402P M1-PK structure is nearly identical with the wild-type structure within experimental error, significant differences in the conformation of the backbone are found at the site of mutation along the Y interface. In addition, there is a significant change along the Z interface, namely, a loss of an inter-subunit salt-bridge between Asp177 of domain B and Arg341 of domain A of the opposing subunit. Concurrent with the loss of the salt-bridge is an increase in the degree of rotational flexibility of domain B that constitutes the active site. Comparison of previous PK structures shows a correlation between an increase in this domain movement with the loss of the Asp177: Arg341 salt-bridge. These results identify the structural linkages between the Y and Z interfaces in regulating the interconversion of conformational states of rabbit M1-PK.
Asunto(s)
Riñón/enzimología , Músculo Esquelético/enzimología , Piruvato Quinasa/química , Piruvato Quinasa/metabolismo , Regulación Alostérica , Sustitución de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Fructosadifosfatos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Mutación , Especificidad de Órganos , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína , Piruvato Quinasa/genética , Conejos , Electricidad Estática , Relación Estructura-ActividadRESUMEN
Despite extensive investigations, the physiological role of the polyol pathway enzyme-aldose reductase (AR) remains obscure. While the enzyme reduces glucose in vivo and in vitro, kinetic and structural studies indicate inefficient carbohydrate binding to the active site of the enzyme. The active site is lined by hydrophobic residues and appears more compatible with the binding of medium- to long-chain aliphatic aldehydes or hydrophobic aromatic aldehydes. In addition, our recent studies show that glutathione (GS) conjugates are also reduced efficiently by the enzyme. For instance, the GS conjugate of acrolein is reduced with a catalytic efficiency 1000-fold higher than the parent aldehyde, indicating specific recognition of glutathione by the active site residues of AR. An increase in the catalytic efficiency upon glutathiolation was also observed with trans-2-nonenal, trans-2-hexenal and trans, trans-2,4-decadienal, establishing that enhancement of catalytic efficiency was specifically due to the glutathione backbone and not specific to the aldehyde. Structure-activity relationships with substitution or deletion of amino acids of GSH indicated specific interactions of the active site with gamma-Glu1 and Cys of GSH. Molecular modeling revealed that the glutathione-propanal conjugate could bind in two distinct orientations. In orientation 1, gamma-Glu1 of the conjugate interacts with Trp20, Lys21 and Val47, and Gly3 interacts with Ser302 and Leu301, whereas in orientation 2, the molecule is inverted with gamma-Glu1 interacting with Ser302, and Leu301. Taken together, these data suggest that glutathiolation of aldehydes enhances their compatibility with the AR active site, which may be of physiological significance in detoxification of endogenous and xenobiotic aldehydes.
Asunto(s)
Aldehído Reductasa/química , Aldehído Reductasa/metabolismo , Glutatión/metabolismo , Aldehídos/química , Aldehídos/metabolismo , Animales , Dominio Catalítico , Glutatión/análogos & derivados , Glutatión/química , Humanos , Hiperglucemia/metabolismo , Técnicas In Vitro , Cinética , Modelos Biológicos , Modelos Moleculares , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
The molecular determinants responsible for flavivirus host cell binding and tissue tropism are largely unknown, although domain III of the envelope protein has been implicated in these functions. We examined the solution properties and antagonist activity of Langat virus domain III. Our results suggest that domain III adopts a stably folded structure that can mediate binding of tick-borne flaviviruses but not mosquito-borne flaviviruses to their target cells. Three clusters of phylogenetically conserved residues are identified that may be responsible for the vector-specific antagonist activity of domain III.
Asunto(s)
Virus de la Encefalitis Transmitidos por Garrapatas/efectos de los fármacos , Receptores Virales/antagonistas & inhibidores , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/farmacología , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops , Dicroismo Circular , Secuencia Conservada , Virus de la Encefalitis Transmitidos por Garrapatas/metabolismo , Flavivirus/efectos de los fármacos , Flavivirus/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Estructura Terciaria de Proteína , Receptores Virales/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , Células Vero , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Little is known about the assembly pathway and structure of hepatitis C virus (HCV) since insufficient quantities of purified virus are available for detailed biophysical and structural studies. Here, we show that bacterially expressed HCV core proteins can efficiently self-assemble in vitro into nucleocapsid-like particles. These particles have a regular, spherical morphology with a modal distribution of diameters of approximately 60 nm. Self-assembly of nucleocapsid-like particles requires structured RNA molecules. The 124 N-terminal residues of the core protein are sufficient for self-assembly into nucleocapsid-like particles. Inclusion of the carboxy-terminal domain of the core protein modifies the core assembly pathway such that the resultant particles have an irregular outline. However, these particles are similar in size and shape to those assembled from the 124 N-terminal residues of the core protein. These results provide novel opportunities to delineate protein-protein and protein-RNA interactions critical for HCV assembly, to study the molecular details of HCV assembly, and for performing high-throughput screening of assembly inhibitors.
Asunto(s)
Hepacivirus/fisiología , Proteínas del Núcleo Viral/metabolismo , Ensamble de Virus , Secuencia de Aminoácidos , Secuencia de Bases , Hepacivirus/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas del Núcleo Viral/genética , Virión/metabolismoRESUMEN
Aldose reductase (AR), a member of the aldo-keto reductase superfamily, has been implicated in the etiology of secondary diabetic complications. However, the physiological functions of AR under euglycemic conditions remain unclear. We have recently demonstrated that, in intact heart, AR catalyzes the reduction of the glutathione conjugate of the lipid peroxidation product 4-hydroxy-trans-2-nonenal (Srivastava, S., Chandra, A., Wang, L., Seifert, W. E., Jr., DaGue, B. B., Ansari, N. H., Srivastava, S. K., and Bhatnagar, A. (1998) J. Biol. Chem. 273, 10893-10900), consistent with a possible role of AR in the metabolism of glutathione conjugates of aldehydes. Herein, we present several lines of evidence suggesting that the active site of AR forms a specific glutathione-binding domain. The catalytic efficiency of AR in the reduction of the glutathione conjugates of acrolein, trans-2-hexenal, trans-2-nonenal, and trans,trans-2,4-decadienal was 4-1000-fold higher than for the corresponding free alkanal. Alterations in the structure of glutathione diminished the catalytic efficiency in the reduction of the acrolein adduct, consistent with the presence of specific interactions between the amino acid residues of glutathione and the AR active site. In addition, non-aldehydic conjugates of glutathione or glutathione analogs displayed active-site inhibition. Molecular dynamics calculations suggest that the conjugate adopts a specific low energy configuration at the active site, indicating selective binding. These observations support an important role of AR in the metabolism of glutathione conjugates of endogenous and xenobiotic aldehydes and demonstrate, for the first time, efficient binding of glutathione conjugates to an aldo-keto reductase.
Asunto(s)
Aldehído Reductasa/química , Aldehído Reductasa/metabolismo , Glutatión/análogos & derivados , Glutatión/metabolismo , Oligopéptidos/metabolismo , Aldehídos/metabolismo , Sitios de Unión , Femenino , Glutatión/química , Humanos , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Oligopéptidos/química , Placenta/enzimología , Embarazo , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Programas Informáticos , Espectrometría de Masa de Ion Secundario , Electricidad Estática , TermodinámicaRESUMEN
Endemic/epidemic dengue viruses (DEN) that are transmitted among humans by the mosquito vectors Aedes aegypti and Aedes albopictus are hypothesized to have evolved from sylvatic DEN strains that are transmitted among nonhuman primates in West Africa and Malaysia by other Aedes mosquitoes. We tested this hypothesis with phylogenetic studies using envelope protein gene sequences of both endemic/epidemic and sylvatic strains. The basal position of sylvatic lineages of DEN-1, -2, and -4 suggested that the endemic/epidemic lineages of these three DEN serotypes evolved independently from sylvatic progenitors. Time estimates for evolution of the endemic/epidemic forms ranged from 100 to 1,500 years ago, and the evolution of endemic/epidemic forms represents relatively recent events in the history of DEN evolution. Analysis of envelope protein amino acid changes predicted to have accompanied endemic/epidemic emergence suggested a role for domain III in adaptation to new mosquito and/or human hosts.
Asunto(s)
Evolución Biológica , Virus del Dengue/genética , Secuencia de Bases , Cartilla de ADN , Virus del Dengue/clasificación , Insectos Vectores , Modelos Moleculares , Filogenia , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Binding of yellow fever virus wild-type strains Asibi and French viscerotropic virus and vaccine strains 17D and FNV to monkey brain and monkey liver cell membrane receptor preparations (MRPs) was investigated. Only FNV bound to monkey brain MRPs, while French viscerotropic virus, Asibi, and FNV all bound to monkey liver MRPs. Four monkey brain and two mouse brain MRP escape (MRP(R)) variants of FNV were selected at pH 7.6 and 6.0. Three monkey brain MRP(R) variants selected at pH 7.6 each had only one amino acid substitution in the envelope (E) protein in domain II (E-237, E-260, or E274) and were significantly attenuated in mice following intracerebral inoculation. Two of the variants were tested in monkeys and retained parental neurotropism following intracerebral inoculation at the dose tested. We speculate that this region of domain II is involved in binding of FNV E protein to monkey brain and is, in part, responsible for the enhanced neurotropism of FNV for monkeys. A monkey brain MRP(R) variant selected at pH 6.0 and two mouse brain MRP(R) variants selected at pH 7.6 were less attenuated in mice, and each had an amino acid substitution in the transmembrane region of the E protein (E-457 or E-458).
Asunto(s)
Encéfalo/virología , Receptores Virales/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Vacunas Virales/metabolismo , Virus de la Fiebre Amarilla/metabolismo , Animales , Encéfalo/metabolismo , Chlorocebus aethiops , Variación Genética , Macaca fascicularis , Ratones , Conformación Proteica , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Vacunas Virales/genética , Virus de la Fiebre Amarilla/genéticaRESUMEN
Six passages of the mosquito-borne flavivirus yellow fever (YF) wild-type strain Asibi in HeLa cells attenuated the virus for monkeys and newborn mice and resulted in loss of mosquito competence. Attenuation after the passage in HeLa cells was not unique to YF virus strain Asibi as demonstrated by the HeLa passage attenuation of wild-type YF virus strain French viscerotropic virus and YF vaccine virus 17D-204 for newborn mice. In contrast, wild-type strain Dakar 1279 and the French neurotropic vaccine virus remained virulent for newborn mice after six passages in HeLa cells. Thus not all strains of YF virus can be attenuated by passage in HeLa cells. Attenuation of YF virus strains Asibi and French viscerotropic virus was accompanied by alterations in the antigenic and biological properties of the viruses, including changes to envelope protein epitopes. Attenuation for newborn mice was coincidental with the acquisition by the HeLa-passaged viruses of the vaccine-specific envelope protein epitope recognized by monoclonal antibody H5. This suggests that this conformational change may play a role in the attenuation process. Wild-type Dakar 1279, which remained virulent for newborn mice after passage in HeLa cells, retained its wild-type antigenic character. The genome of Asibi HeLa p6 virus differed from wild-type Asibi virus by 29 nucleotides that encoded 10 amino acid substitutions: 5 in the envelope protein, 1 in NS2A, 3 in NS4B, and 1 in NS5. The substitution at NS4B-95 is seen in three different attenuation processes of wild-type YF virus, leading us to speculate that it is involved in the attenuation of virulence of wild-type strain Asibi.
Asunto(s)
Células HeLa/virología , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/fisiología , Virus de la Fiebre Amarilla/patogenicidad , Animales , Humanos , Ratones , Especificidad de la Especie , Proteínas del Envoltorio Viral/fisiología , Virulencia , Replicación ViralRESUMEN
Aldose reductase (AR) is a member of the aldo-keto reductase superfamily. Due to its ability to catalyze the formation of sorbitol from glucose during hyperglycemic and hypertonic stress, the aldose-reducing property of AR has been accepted as its main physiological and pathological function. Nonetheless, AR is a poor catalyst for glucose reduction and displays active-site properties unexpected of a carbohydrate-binding protein. We, therefore, examined the catalytic properties of AR with a series of naturally occurring aldehydes, compatible in their hydrophobicity to the large apolar active site of the enzyme. Our results show that recombinant human AR is an efficient catalyst for the reduction of medium- to long-chain unbranched saturated and unsaturated aldehydes. The enzyme displayed selective preference for saturated aldehydes, such as hexanal, and unsaturated aldehydes, such as trans-2-octenal and nonenal as well as their 4-hydroxy derivatives. Short-chain aldehydes such as propanal and acrolein were reduced less efficiently. Branched derivatives of acrolein or its glutathione conjugate (GS-propanal) were, however, reduced with high efficiency. In the absence of NADPH, the alpha, beta unsaturated aldehydes caused covalent modification of the enzyme. On the basis of electrospray mass spectrometric analysis of the wild-type and site-directed mutants of AR (in which the solvent exposed cysteines were individually replaced with serine), the site of modification was identified to be the active-site residue, Cys 298. The unsaturated aldehydes, however, did not modify the enzyme bound to NADPH and did not inactivate the enzyme during catalysis. Modeling studies indicate that the large hydrophobic active site of AR can accommodate a large number of aldehydes without changes in the structure of the binding site or movement of side chains. High hydrophobicity due to long alkyl chains or apolar substituents appears to stabilize the interaction of the aldehyde substrates with the enzyme. Apparently, such hydrophobic interactions provide substrate selectivity and catalytic efficiency of the order achievable by hydrogen bonding. Since several of the aldehydes reduced by AR are either environmental and pharmacological pollutants or products of lipid peroxidation, the present studies provide the basis of future investigations on the role of AR in regulating aldehyde metabolism particularly under pathological states associated with oxidative stress and/or aldehyde toxicity.
Asunto(s)
Aldehído Reductasa/química , Aldehído Reductasa/metabolismo , Aldehídos/química , Aldehídos/metabolismo , Catálisis , Simulación por Computador , Humanos , Cinética , Espectrometría de Masas , Modelos Moleculares , Oxidación-Reducción , Especificidad por SustratoRESUMEN
The heterogeneous nature of the yellow fever (YF) 17D-204 vaccine virus population was exploited in this study to isolate virus variants able to escape neutralization by the 17D-204 vaccine-specific MAb 864. The conformational change on the virus surface that resulted in the loss of the MAb 864-defined epitope was effected in each variant by a single amino acid mutation in the envelope (E) protein at either position E-305 or E-325. Interestingly, both positions were mutated during attenuation of the 17D-204 vaccine substrain from the wildtype Asibi strain. The mutations in several of the variants represented reversion to the wildtype Asibi virus sequence consistent with loss of a 17D-204 substrain-specific epitope. The majority of the variant viruses were shown to have altered mouse neurovirulence phenotypes, ranging from complete avirulence through to increased virulence. The avirulent variants are the first flavivirus MAb-neutralization-resistant variants to be attenuated for neurovirulence in the adult mouse model. Overall, the results indicate that the E protein epitope recognized by MAb 864 defines a functionally important region that encodes major molecular determinants of YF virus pathogenesis in vivo.
Asunto(s)
Epítopos de Linfocito B/genética , Mutación Puntual , Proteínas del Envoltorio Viral/genética , Vacunas Virales , Virus de la Fiebre Amarilla/genética , Virus de la Fiebre Amarilla/patogenicidad , Animales , Encéfalo/patología , Encéfalo/virología , Chlorocebus aethiops , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Femenino , Inmunohistoquímica , Ratones , Conformación Proteica , Análisis de Secuencia de ADN , Células Vero , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/genética , Vacunas Virales/inmunología , Virulencia , Replicación Viral , Fiebre Amarilla/patología , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/inmunología , Virus de la Fiebre Amarilla/fisiologíaRESUMEN
Previous x-ray studies (2.8-A resolution) on crystals of tobacco mosaic virus coat protein grown from solutions containing high salt have characterized the structure of the protein aggregate as a dimer of a bilayered cylindrical disk formed by 34 chemically identical subunits. We have determined the crystal structure of the disk aggregate at 2.4-A resolution using x-ray diffraction from crystals maintained at cryogenic temperatures. Two regions of interest have been extensively refined. First, residues of the low-radius loop region, which were not modeled previously, have been traced completely in our electron density maps. Similar to the structure observed in the virus, the right radial helix in each protomer ends around residue 87, after which the protein chain forms an extended chain that extends to the left radial helix. The left radial helix appears as a long alpha-helix with high temperature factors for the main-chain atoms in the inner portion. The side-chain atoms in this region (residues 90-110) are not visible in the electron density maps and are assumed to be disordered. Second, interactions between subunits in the symmetry-related central A pair have been determined. No direct protein-protein interactions are observed in the major overlap region between these subunits; all interactions are mediated by two layers of ordered solvent molecules. The current structure emphasizes the importance of water in biological macromolecular assemblies.
Asunto(s)
Cápside/química , Estructura Secundaria de Proteína , Virus del Mosaico del Tabaco/química , Secuencia de Aminoácidos , Cápside/aislamiento & purificación , Simulación por Computador , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Plantas Tóxicas , Nicotiana/virologíaRESUMEN
X-ray diffraction data collected at cryogenic temperatures from flash-cooled crystals of influenza virus hemagglutinin show improvements in both resolution and quality relative to data collected at 277 K. These improvements are dramatic for flash-cooled hemagglutinin crystals irradiated with X-rays from a synchrotron source. At the Cornell High Energy Synchrotron Source flash-cooled hemagglutinin crystals diffracted at least 0.9 A farther than hemagglutinin crystals at ambient temperatures. Radiation damage in the flash-cooled crystals is reduced, making it possible to collect a complete data set from a single hemagglutinin crystal. However, radiation damage is not eliminated in the flash-cooled crystal. As a result the quality of X-ray data can be significantly degraded during long exposure times at a synchrotron source.
RESUMEN
BACKGROUND: The first step in influenza A virus infection involves attachment to cells through binding of viral hemagglutinin to cell-surface receptors containing alpha-5-N-acetylneuraminic acid (sialic acid). The structures of soluble hemagglutinin in isolation and in complex with several low-affinity receptor analogs have been solved previously to approximately 3A resolution. To design effective, and possibly therapeutic, inhibitors of viral attachment we have determined the structure of hemagglutinin in complex with four high-affinity (10-fold to 100-fold higher affinity) sialic acid analogs at higher resolution. RESULTS: In each crystal structure the sialic acid moiety is equivalently positioned in the receptor binding site but the substituent groups that differentiate the high-affinity analogs from each other interact with hydrophobic patches and polar residues adjacent to the binding site. Re-examination of the receptor binding site at 2.15A resolution reveals several hydrophilic pockets and an apolar channel that adjoin the receptor binding site. CONCLUSIONS: The interactions observed in the structures of soluble hemagglutinin in complex with receptor analogs suggest explanations for the observed affinities of the analogs, designs for potential sialic acid analogs with even higher affinities, and ideas both for inhibiting membrane fusion and for circumventing evasion of inhibition by antigenic variation.
Asunto(s)
Hemaglutininas Virales/química , Receptores de Superficie Celular/química , Receptores Virales/química , Ácidos Siálicos/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Glicoproteínas Hemaglutininas del Virus de la Influenza , Hemaglutininas Virales/metabolismo , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Ácidos Siálicos/metabolismo , Especificidad de la EspecieRESUMEN
An atomic model of the sickle hemoglobin (HbS) fiber was synthesized by combining the molecular coordinates of the fiber (obtained from electron microscopy) with atomic coordinates of the sickle hemoglobin double strand (obtained from X-ray crystallography). The model is stereochemically acceptable. The majority of polymerization-sensitive HbS mutants are located at fiber contact sites and the majority of the mutants that do not affect polymerization are not located at contact sites. The residues at intermolecular contacts in the fiber model are reported. We have searched the coordinate space in the vicinity of the EM reconstructions to find models with alternative sets of coordinates that satisfy the mutant data, contain 5-A contacts between double strands, and are stereochemically acceptable. This involved a systematic examination over 297 different models. The alternative fiber models were generated with a range of fiber pitch, double-strand positions, and double-strand polarity. Models which had unacceptably close contacts between atoms, failed to satisfy the mutant data, or did not have 5-A contacts between double strands were considered unacceptable. None of the acceptable alternative fiber models improved the agreement between the polymerization behavior of HbS mutants and their contact site location. However, several models could account for the polymerization data equally well. Residue locations for single-site HbS mutations that could discriminate between alternative fiber models are proposed. The twist of HbS fibers varies in an apparent random manner with an average rotation of 7.8 +/- 2.5 degrees per molecule and a maximum rotation of 16 degrees per molecule. The number of interdouble-strand contacts as a function of fiber twist shows a broad maximum around 9 degrees and may account for the observed range of fiber pitch. This study shows that the upper limit on the fiber twist could result from a loss of axial contacts and repulsive van der Waals interactions between residues involved in interstrand contacts. The loss of axial contacts limits the radial growth of the fiber. In the appendix we analyze the methodology used by I. Cretegny and S. J. Edelstein [(1993) J. Mol. Biol. 230, 733-738] to build a model of the fiber. Our examination reveals shortcomings in the methodology of Cretegny and Edelstein. One result of these shortcomings is that the model synthesized by Cretegny and Edelstein is not stereochemically acceptable because it gives rise to a large number of excessively close (less than 1.4 A) atom-atom contacts, suggesting interpenetration of the molecular envelopes.
Asunto(s)
Hemoglobina Falciforme/química , Secuencia de Aminoácidos , Anemia de Células Falciformes/sangre , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/terapia , Sitios de Unión , Biopolímeros/química , Cristalografía por Rayos X , Hemoglobina Falciforme/genética , Humanos , Microscopía Electrónica , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Conformación Proteica , TermodinámicaRESUMEN
By combining X-ray crystallographic co-ordinates of sickle hemoglobin (HbS) molecules with three-dimensional reconstructions of electron micrographs of HbS fibers we have synthesized a model for the structure of the clinically relevant HbS fiber. This model largely accounts for the action of 55 point mutations of HbS whose effect on fiber formation has been studied. In addition, it predicts locations at which additional point mutations are likely to affect fiber formation. The number of intermolecular axial contacts decreases with radius until, at the periphery of the fiber, there are essentially no axial contacts. We suggest that this observation accounts for the limited radial growth of the HbS fiber and that a similar mechanism may be a factor in limiting the size of other helical particles. The methodology for the synthesis of the fiber model is applicable to other systems in which X-ray crystallographic and electron microscopic data are available.