RESUMEN
A novel technique is described, using serial photography of the gut contents of transparent living larval fishes, to generate individual gut evacuation time series. This technique was applied to Atlantic mackerel Scomber scombrus larvae to compare three widely used models of gut evacuation: linear, exponential and square-root. Regression r(2) for the exponential model exceeded those for the linear and square root models in 20 of 21 time series, strongly supporting the exponential model. At the initial gut fullness for each time series, total gut evacuation rates calculated with the exponential model averaged 2.2 and 1.3 times greater than those calculated with the linear and square-root models, respectively, and would produce correspondingly higher estimates of feeding rates for field-collected larvae with similar levels of gut fullness. The results highlight the importance of choosing the appropriate evacuation model in feeding studies, particularly those intended to examine short-term changes in larval fish feeding rates, a contributing factor to the highly variable yearly recruitment of many marine fish species.
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Tracto Gastrointestinal/fisiología , Biología Marina/métodos , Modelos Biológicos , Perciformes/fisiología , Fotograbar , Animales , Contenido DigestivoRESUMEN
A backbone-dependent rotamer library for amino acid side-chains is developed and used for constructing protein side-chain conformations from the main-chain co-ordinates. The rotamer library is obtained from 132 protein chains in the Brookhaven Protein Database. A grid of 20 degrees by 20 degrees blocks for the main-chain angles phi, psi is used in the rotamer library. Significant correlations are found between side-chain dihedral angle probabilities and backbone phi, psi values. These probabilities are used to place the side-chains on the known backbone in test applications for six proteins for which high-resolution crystal structures are available. A minimization scheme is used to reorient side-chains that conflict with the backbone or other side-chains after the initial placement. The initial placement yields 59% of both chi 1 and chi 2 values in the correct position (to within 40 degrees) for thermolysin to 81% for crambin. After refinement the values range from 61% (lysozyme) to 89% (crambin). It is evident from the results that a single protein does not adequately test a prediction scheme. The computation time required by the method scales linearly with the number of side-chains. An initial prediction from the library takes only a few seconds of computer time, while the iterative refinement takes on the order of hours. The method is automated and can easily be applied to aid experimental side-chain determinations and homology modeling. The high degree of correlation between backbone and side-chain conformations may introduce a simplification in the protein folding process by reducing the available conformational space.
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Bases de Datos Factuales , Conformación Proteica , Proteínas/química , Programas Informáticos , Secuencia de Aminoácidos , Enzimas/química , Modelos Moleculares , Datos de Secuencia Molecular , Probabilidad , Difracción de Rayos X/métodosRESUMEN
Modeling by homology is the most accurate computational method for translating an amino acid sequence into a protein structure. Homology modeling can be divided into two sub-problems, placing the polypeptide backbone and adding side-chains. We present a method for rapidly predicting the conformations of protein side-chains, starting from main-chain coordinates alone. The method involves using fewer than ten rotamers per residue from a backbone-dependent rotamer library and a search to remove steric conflicts. The method is initially tested on 299 high resolution crystal structures by rebuilding side-chains onto the experimentally determined backbone structures. A total of 77% of chi1 and 66% of chi(1 + 2) dihedral angles are predicted within 40 degrees of their crystal structure values. We then tested the method on the entire database of known structures in the Protein Data Bank. The predictive accuracy of the algorithm was strongly correlated with the resolution of the structures. In an effort to simulate a realistic homology modeling problem, 9424 homology models were created using three different modeling strategies. For prediction purposes, pairs of structures were identified which shared between 30% and 90% sequence identity. One strategy results in 82% of chi1 and 72% chi(1 + 2) dihedral angles predicted within 40 degrees of the target crystal structure values, suggesting that movements of the backbone associated with this degree of sequence identity are not large enough to disrupt the predictive ability of our method for non-native backbones. These results compared favorably with existing methods over a comprehensive data set.
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Algoritmos , Simulación por Computador , Modelos Moleculares , Conformación Proteica , Homología de Secuencia de Aminoácido , Aminoácidos/química , Bases de Datos Factuales , Reproducibilidad de los ResultadosRESUMEN
The enzyme BACE (beta-site APP-cleaving enzyme) has recently been identified as the beta-secretase that cleaves the amyloid precursor protein (APP) to produce the N terminus of the Abeta peptide found in plaques in the brains of Alzheimer's disease patients. BACE is an aspartic protease similar to pepsin and renin. Comparative modeling of the three-dimensional structure of BACE in complex with its substrate shows that several residues confer specificity of the enzyme for APP. In particular, Arg296 forms a salt-bridge with the P1' Asp of the APP substrate, explaining the unusual preference of BACE among aspartic proteases for a P1' residue that is negatively charged. Several hydrophobic residues in the enzyme form a pocket for the P1 hydrophobic residue (Met in wild-type APP and Leu in APP with the "Swedish mutation" associated with early-onset of Alzheimer's disease). Inhibitors that can bind to the BACE active site may prove useful for drugs to treat and prevent Alzheimer's disease.
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Enfermedad de Alzheimer/enzimología , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/química , Ácido Aspártico Endopeptidasas/metabolismo , Modelos Moleculares , Enfermedad de Alzheimer/genética , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/química , Animales , Arginina/metabolismo , Ácido Aspártico/metabolismo , Ácido Aspártico Endopeptidasas/genética , Sitios de Unión , Cristalografía por Rayos X , Endopeptidasas , Humanos , Enlace de Hidrógeno , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Alineación de Secuencia , Electricidad Estática , Especificidad por SustratoRESUMEN
We present a Bayesian statistical analysis of the conformations of side chains in proteins from the Protein Data Bank. This is an extension of the backbone-dependent rotamer library, and includes rotamer populations and average chi angles for a full range of phi, psi values. The Bayesian analysis used here provides a rigorous statistical method for taking account of varying amounts of data. Bayesian statistics requires the assumption of a prior distribution for parameters over their range of possible values. This prior distribution can be derived from previous data or from pooling some of the present data. The prior distribution is combined with the data to form the posterior distribution, which is a compromise between the prior distribution and the data. For the chi 2, chi 3, and chi 4 rotamer prior distributions, we assume that the probability of each rotamer type is dependent only on the previous chi rotamer in the chain. For the backbone-dependence of the chi 1 rotamers, we derive prior distributions from the product of the phi-dependent and psi-dependent probabilities. Molecular mechanics calculations with the CHARMM22 potential show a strong similarity with the experimental distributions, indicating that proteins attain their lowest energy rotamers with respect to local backbone-side-chain interactions. The new library is suitable for use in homology modeling, protein folding simulations, and the refinement of X-ray and NMR structures.
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Teorema de Bayes , Conformación Proteica , Modelos Químicos , Modelos MolecularesRESUMEN
New protein parameters are reported for the all-atom empirical energy function in the CHARMM program. The parameter evaluation was based on a self-consistent approach designed to achieve a balance between the internal (bonding) and interaction (nonbonding) terms of the force field and among the solvent-solvent, solvent-solute, and solute-solute interactions. Optimization of the internal parameters used experimental gas-phase geometries, vibrational spectra, and torsional energy surfaces supplemented with ab initio results. The peptide backbone bonding parameters were optimized with respect to data for N-methylacetamide and the alanine dipeptide. The interaction parameters, particularly the atomic charges, were determined by fitting ab initio interaction energies and geometries of complexes between water and model compounds that represented the backbone and the various side chains. In addition, dipole moments, experimental heats and free energies of vaporization, solvation and sublimation, molecular volumes, and crystal pressures and structures were used in the optimization. The resulting protein parameters were tested by applying them to noncyclic tripeptide crystals, cyclic peptide crystals, and the proteins crambin, bovine pancreatic trypsin inhibitor, and carbonmonoxy myoglobin in vacuo and in crystals. A detailed analysis of the relationship between the alanine dipeptide potential energy surface and calculated protein φ, χ angles was made and used in optimizing the peptide group torsional parameters. The results demonstrate that use of ab initio structural and energetic data by themselves are not sufficient to obtain an adequate backbone representation for peptides and proteins in solution and in crystals. Extensive comparisons between molecular dynamics simulations and experimental data for polypeptides and proteins were performed for both structural and dynamic properties. Energy minimization and dynamics simulations for crystals demonstrate that the latter are needed to obtain meaningful comparisons with experimental crystal structures. The presented parameters, in combination with the previously published CHARMM all-atom parameters for nucleic acids and lipids, provide a consistent set for condensed-phase simulations of a wide variety of molecules of biological interest.
RESUMEN
We present results of comparative modeling on 11 targets from the CASP3 experiment. Our methods comprise the following steps: first, PSI-BLAST is used to find homologues of the target sequence in the nonredundant GenBank protein sequence database; second, after several iterations of PSI-BLAST, the resulting profile or position-specific similarity matrix is used to search a database of Protein Databank (PDB) sequences; third, from the list of hits resulting from the PDB search, a parent structure is chosen on the basis of the quality of the alignment and the quality of the experimental structure; fourth, this alignment is adjusted manually whenever insertions or deletions take place in secondary structure regions of the parent; fifth, the backbone is modeled from the parent structure and the alignment; and finally, the program SCWRL is used to replace nonconserved side chains onto the parent backbone given the target sequence. For comparison, we also produced structural models from the unaltered PSI-BLAST alignment, from an alignment from the nonprofile version of BLAST, and from the global sequence alignment program CLUSTAL W. Our results indicate that PSI-BLAST produced considerably better alignments than would be possible with either global or local pairwise sequence alignment algorithms and that manual adjustments were helpful. SCWRL, which uses a backbone-dependent rotamer library to predict side-chain conformations, did well in comparison with other methods used in CASP3.
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Algoritmos , Modelos Moleculares , Proteínas/química , Bases de Datos Factuales , Conformación Proteica , Alineación de SecuenciaRESUMEN
Amino acids have sidechain rotamer preferences dependent on the backbone dihedral angles phi and psi. These preferences provide a method for rapid structure prediction which is a significant improvement over backbone-independent rotamer libraries. We demonstrate here that simple arguments based on conformational analysis can account for many of the features of the observed backbone dependence of the sidechain rotamers. Steric repulsions corresponding to the 'butane' and 'syn-pentane' effects make certain conformers rare, as has been observed experimentally.
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Conformación Proteica , Aminoácidos/química , Hidrocarburos/química , Modelos Moleculares , Conformación Molecular , RotaciónRESUMEN
The first available genome of a multicellular organism, C. elegans, was used as a test case for protein fold assignment using PSI-BLAST, followed by rational structure modeling and interpretation of experimental mutagenesis data in the context of collaboration with biologists. Similar results are demonstrated for human disease proteins with known polymorphisms.
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Caenorhabditis elegans/genética , Genoma , Modelos Moleculares , Pliegue de Proteína , Proteínas , Animales , HumanosRESUMEN
Sequence alignment programs such as BLAST and PSI-BLAST are used routinely in pairwise, profile-based, or intermediate-sequence-search (ISS) methods to detect remote homologies for the purposes of fold assignment and comparative modeling. Yet, the sequence alignment quality of these methods at low sequence identity is not known. We have used the CE structure alignment program (Shindyalov and Bourne, Prot Eng 1998;11:739) to derive sequence alignments for all superfamily and family-level related proteins in the SCOP domain database. CE aligns structures and their sequences based on distances within each protein, rather than on interprotein distances. We compared BLAST, PSI-BLAST, CLUSTALW, and ISS alignments with the CE structural alignments. We found that global alignments with CLUSTALW were very poor at low sequence identity (<25%), as judged by the CE alignments. We used PSI-BLAST to search the nonredundant sequence database (nr) with every sequence in SCOP using up to four iterations. The resulting matrix was used to search a database of SCOP sequences. PSI-BLAST is only slightly better than BLAST in alignment accuracy on a per-residue basis, but PSI-BLAST matrix alignments are much longer than BLAST's, and so align correctly a larger fraction of the total number of aligned residues in the structure alignments. Any two SCOP sequences in the same superfamily that shared a hit or hits in the nr PSI-BLAST searches were identified as linked by the shared intermediate sequence. We examined the quality of the longest SCOP-query/ SCOP-hit alignment via an intermediate sequence, and found that ISS produced longer alignments than PSI-BLAST searches alone, of nearly comparable per-residue quality. At 10-15% sequence identity, BLAST correctly aligns 28%, PSI-BLAST 40%, and ISS 46% of residues according to the structure alignments. We also compared CE structure alignments with FSSP structure alignments generated by the DALI program. In contrast to the sequence methods, CE and structure alignments from the FSSP database identically align 75% of residue pairs at the 10-15% level of sequence identity, indicating that there is substantial room for improvement in these sequence alignment methods. BLAST produced alignments for 8% of the 10,665 nonimmunoglobulin SCOP superfamily sequence pairs (nearly all <25% sequence identity), PSI-BLAST matched 17% and the double-PSI-BLAST ISS method aligned 38% with E-values <10.0. The results indicate that intermediate sequences may be useful not only in fold assignment but also in achieving more complete sequence alignments for comparative modeling.
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Algoritmos , Proteínas/química , Alineación de Secuencia/métodos , Bases de Datos Factuales , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Several structural features of the cytoplasmic domain of CD4 including phosphorylation of Ser-408 have been shown to be important in its endocytosis (Shin, J., Doyle, C., Yang, Z., Kappes, D., and Strominger, J.L. (1990) EMBO J. 9, 425-434). A series of cytoplasmic domain truncations have now indicated that the membrane proximal region of the cytoplasmic domain from Arg-396 to Lys-417 is sufficient for phorbol ester-induced internalization; this segment is predicted to be an alpha-helix. The severe impairment of endocytosis resulting from the mutation Ser-408 to Ala-408 is largely restored by a compensating mutation Ala-404 to Ser-404; phosphorylation of Ser-404 has been directly demonstrated. Furthermore, mutation of Met-407, Ile-410, Leu-413, or Leu-414 to a hydrophilic residue eliminated CD4 endocytosis as did domain truncation at Arg-412. Ser-408 was normally phosphorylated in all of these mutants, suggesting that other residues in this region, including the four hydrophobic amino acids, are also required for CD4 endocytosis. Immunofluorescence microscopy following staining of intact and permeabilized cells showed that all endocytosis defective mutants indeed remained on the cell surface even after phorbol ester treatment, while wild type CD4 was endocytosed and degraded in lysosomes. These data indicate that endocytosis requiring residues 397-417 and binding of lymphocyte tyrosine kinase at residues 417-429 are functions of independent segments of the cytoplasmic region and lead to a hypothesis regarding some features of the endocytic process.
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Antígenos CD4/metabolismo , Regulación hacia Abajo , Secuencia de Aminoácidos , Antígenos CD4/genética , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/enzimología , Electroforesis en Gel de Poliacrilamida , Endocitosis , Humanos , Microscopía Fluorescente , Datos de Secuencia Molecular , Mutación , Fosforilación , Pruebas de Precipitina , Conformación Proteica , Proteínas Tirosina Quinasas/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
Human porphobilinogen synthase (PBGS) is a main target in lead poisoning. Human PBGS purifies with eight Zn(II) per homo-octamer; four ZnA have predominantly nonsulfur ligands, and four ZnB have predominantly sulfur ligands. Only four Zn(II) are required for activity. To better elucidate the roles of Zn(II) and Pb(II), we produced human PBGS mutants that are designed to lack either the ZnA or ZnB sites. These proteins, MinusZnA (H131A, C223A) and MinusZnB (C122A, C124A, C132A), each become purified with four Zn(II) per octamer, thus confirming an asymmetry in the human PBGS structure. MinusZnA is fully active, whereas MinusZnB is far less active, verifying an important catalytic role for ZnB and the removed cysteine residues. Kinetic properties of the mutants and wild type proteins are described. Comparison of Pb(II) inhibition of the mutants shows that ligands to both ZnA and ZnB interact with Pb(II). The ZnB ligands preferentially interact with Pb(II). At least one ZnA ligand is responsible for the slow tight binding behavior of Pb(II). The data support a novel model where a high affinity lead site is a hybrid of the ZnA and ZnB sites. We propose that the lone electron pair of Pb(II) precludes Pb(II) to function in PBGS catalysis.
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Plomo/farmacología , Porfobilinógeno Sintasa/antagonistas & inhibidores , Porfobilinógeno Sintasa/química , Zinc/metabolismo , Sustitución de Aminoácidos , Sitios de Unión , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Porfobilinógeno Sintasa/genética , Conformación Proteica , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Eliminación de SecuenciaRESUMEN
Human cystathionine beta--synthase (CBS) is an S-adenosylmethionine-regulated enzyme that plays a key role in the metabolism of homocysteine. Mutations in CBS are known to cause homocystinuria, an inborn error in metabolism. We previously developed a yeast functional assay for CBS and used it to characterize mutations found in homocystinuric patients. We discovered that many patient-derived mutations are functionally suppressed by deletion of the C-terminal 142 amino acids, which contain a 53 amino acid motif known as the CBS domain. This domain is found in a wide variety of proteins of diverse biological function. Here we have used a genetic screen to identify missense mutations in the C-terminal region of CBS that can suppress the most common patient mutation, I278T. Seven suppressor mutations were identified, four of which map to the CBS domain. When combined in cis with another pathogenic mutation, V168M, six of seven of the suppressor mutations rescued the yeast phenotype. Enzyme activity analyses indicate that the suppressors restore activity from <2% to 17--64% of the wild-type levels. Analysis of the suppressor mutations in the absence of the pathogenic mutation shows that six of the seven suppressor alleles have lost enzymatic responsiveness to S-adenosylmethionine. Using homology modeling, we show that the suppressor mutations appear to map on one face of the CBS domain. Our results indicate that subtle changes to the C-terminus of CBS can restore activity to mutant proteins and provide a rationale for screening for compounds that can activate mutant CBS alleles.
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Cistationina betasintasa/genética , Regulación Enzimológica de la Expresión Génica , Homocistinuria/genética , Secuencia de Aminoácidos , Cistationina betasintasa/metabolismo , Pruebas Genéticas , Homocistinuria/enzimología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Homología de Secuencia de AminoácidoRESUMEN
UNLABELLED: BeoBLAST is an integrated software package that handles user requests and distributes BLAST and PSI-BLAST searches to nodes of a Beowulf cluster, thus providing a simple way to implement a scalable BLAST system on top of relatively inexpensive computer clusters. Additionally, BeoBLAST offers a number of novel search features through its web interface, including the ability to perform simultaneous searches of multiple databases with multiple queries, and the ability to start a search using the PSSM generated from a previous PSI-BLAST search on a different database. The underlying system can also handle automated querying for high throughput work. AVAILABILITY: Source code is available under the GNU public license at http://bioinformatics.fccc.edu/
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Redes de Comunicación de Computadores , Metodologías Computacionales , Sistemas de Administración de Bases de Datos , Bases de Datos Genéticas , Almacenamiento y Recuperación de la Información/métodos , Internet , National Library of Medicine (U.S.) , Análisis de Secuencia , Estados UnidosRESUMEN
A mutant of CD4 (CD4.Q421stop), in which the cytoplasmic C-terminal 13 amino acids were truncated, was not expressed on the surface of HeLa cells after transfection but was retained in the endoplasmic reticulum (ER). Seven other truncation mutants of CD4 were expressed well on the cell surface, thus suggesting that the C-terminal amino acids of CD4.Q421stop (-Ser-Glu-Lys-Lys-Thr-Cys) may have the sequence information for ER retention. Further mutational study has revealed that two consecutive lysine residues at the third and fourth positions from the C-terminal end are sufficient for ER retention. Lysine at the fourth position, but not at the third position, from the C terminus can be replaced by arginine without disturbing ER retention. Furthermore, two lysine residues at the third and fifth positions from the C terminus also resulted in ER retention. Thus lysine at the third position and a positively charged amino acid either at the fourth or fifth position from the C terminus are sufficient for ER retention of this CD4 mutant, and possibly all transmembrane proteins. In addition to the requirement of specific amino acids at specific positions, the ER retention signal -Lys-Lys-Xaa-Xaa also requires a transmembrane region for function. By contrast -Lys-Asp-Glu-Leu, which targets soluble proteins to the lumen of the ER, does not function in the presence of a transmembrane region.
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Antígenos CD4/genética , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Mutagénesis Sitio-Dirigida , Transducción de Señal , Secuencia de Aminoácidos , Animales , Células HeLa/inmunología , Células HeLa/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Conejos , Ratas , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
Pyrophosphate-dependent phosphofructokinase (PPi-PFK) is the rate-limiting glycolytic enzyme found in the pathogenic protists Entamoeba histolytica, Giardia lamblia, Toxoplasma gondii, Trichomonas vaginalis, and Naegleria fowleri. The enzyme differs significantly from ATP-dependent phosphofructokinases found in humans and as such represents an important drug target. Current therapy for infections caused by these pathogens is inadequate, especially for children, pregnant women, and the immune compromised. The development of more selective, safer agents in imperative, as parasitic infections are currently a significant health threat worldwide and will likely become increasingly common agents of disease in the future. For the purpose of designing drugs to treat parasitic infections, we have constructed a model of PPi-PFK from E. histolytica based on the three-dimensional structure of the ATP-dependent PFK from Bacillus stearothermophilus. The model was used with the computer program Dock 3.5 (University of California, San Francisco) to predict the binding of pyrophosphate and selected bisphosphonates to the enzyme. The predicted drug-enzyme interactions suggested that two of these compounds would be competitive inhibitors of pyrophosphate. These drugs were tested against E. histolytica and inhibited the growth of amebae in vitro. This class of compounds may have broad-spectrum antiparasitic activity and, in the future, may facilitate the treatment of serious parasitic infections.
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Antiprotozoarios/metabolismo , Difosfatos/metabolismo , Difosfonatos/metabolismo , Entamoeba histolytica/enzimología , Ácido Etidrónico/metabolismo , Imidazoles/metabolismo , Fosfofructoquinasa-1/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Simulación por Computador , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Entamoeba histolytica/citología , Entamoeba histolytica/efectos de los fármacos , Modelos Moleculares , Datos de Secuencia Molecular , Fosfofructoquinasa-1/antagonistas & inhibidores , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Ácido ZoledrónicoRESUMEN
With the increase in our understanding of its structure and enzymatic mechanism, HIV-1 integrase (IN) has become a promising target for designing drugs to treat patients with AIDS. To investigate the structure and function of IN, a panel of monoclonal antibodies (mAbs) directed against HIV-1 IN was raised and characterized previously in this laboratory. Among them, mAbs17, -4, and -33 were found to inhibit IN activity in vitro. In this study, we investigated the interaction of N-terminal-specific mAb17 and its isolated Fab fragment with full-length HIV-1 IN(1-288) and its isolated N-terminal, Zn(2+)-binding domain IN(1-49). Our results show that binding of Zn(2+) to IN(1-49) stabilizes the mAb17-IN complex and that dimer dissociation is not required for binding of the Fab. To identify the epitope recognized by mAb17, we developed a protein footprinting technique based on controlled proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Binding was mapped to a region within amino acids Asp(25)-Glu(35). This peptide corresponds to the end of a helix-turn-helix motif in the IN(1-55) NMR structure and contributes to the dimerization of the N-terminal domain. Antibody binding also appears to destabilize the N-terminal helix in this domain. A molecular model of the [IN(1-49)](2).(Fab)(1) complex shows Fab binding across the dimer protein and suggests a potential target for drug design. These data also suggest that mAb17 inhibits integrase activity by blocking critical protein-protein interactions and/or by distorting the orientation of the N-terminal alpha-helix. The relevance of our results to an understanding of IN function is discussed.
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Anticuerpos Monoclonales/química , Integrasa de VIH/química , Integrasa de VIH/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión de Anticuerpos , Dominio Catalítico , Ensayo de Inmunoadsorción Enzimática , VIH-1/enzimología , Secuencias Hélice-Giro-Hélice , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The large form of the hepatitis delta virus (HDV) protein (L) can be isoprenylated near its C terminus, and this modification is considered essential for particle assembly. Using gel electrophoresis, we separated L into two species of similar mobilities. The slower species could be labeled by the incorporation of [(14)C]mevalonolactone and is interpreted to be isoprenylated L (L(i)). In serum particles, infected liver, transfected cells, and assembled particles, 25 to 85% of L was isoprenylated. Isoprenylation was also demonstrated by (14)C incorporation in vitro with a rabbit reticulocyte coupled transcription-translation system. However, the species obtained migrated even slower than that detected by labeling in vivo. Next, in studies of HDV particle assembly in the presence of the surface proteins of human hepatitis B virus, we observed the following. (i) Relative to L, L(i) was preferentially assembled into virus-like particles. (ii) L(i) could coassemble the unmodified L and the small delta protein, S. (iii) In contrast, a form of L with a deletion in the dimerization domain was both isoprenylated and assembled, but it could not support the coassembly of S. Finally, to test the expectation that the isoprenylation of L would increase its hydrophobicity, we applied a phase separation strategy based on micelle formation with the nonionic detergent Triton X-114. We showed the following. (i) The unique C-terminal 19 amino acids present on L relative to S caused a significant increase in the hydrophobicity. (ii) This increase was independent of isoprenylation. (iii) In contrast, other, artificial modifications at either the N or C terminus of S did not increase the hydrophobicity. (iv) The increased hydrophobicity was not sufficient for particle assembly; nevertheless, we speculate that it might facilitate virion assembly.
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Antígenos de la Hepatitis/metabolismo , Virus de la Hepatitis Delta/metabolismo , Secuencia de Aminoácidos , Animales , Electroforesis en Gel de Poliacrilamida , Antígenos de Hepatitis delta , Humanos , Datos de Secuencia Molecular , Conejos , Reticulocitos , Homología de Secuencia de Aminoácido , Células Tumorales CultivadasRESUMEN
The results of the second Critical Assessment of Fully Automated Structure Prediction (CAFASP2) are presented. The goals of CAFASP are to (i) assess the performance of fully automatic web servers for structure prediction, by using the same blind prediction targets as those used at CASP4, (ii) inform the community of users about the capabilities of the servers, (iii) allow human groups participating in CASP to use and analyze the results of the servers while preparing their nonautomated predictions for CASP, and (iv) compare the performance of the automated servers to that of the human-expert groups of CASP. More than 30 servers from around the world participated in CAFASP2, covering all categories of structure prediction. The category with the largest participation was fold recognition, where 24 CAFASP servers filed predictions along with 103 other CASP human groups. The CAFASP evaluation indicated that it is difficult to establish an exact ranking of the servers because the number of prediction targets was relatively small and the differences among many servers were also small. However, roughly a group of five "best" fold recognition servers could be identified. The CASP evaluation identified the same group of top servers albeit with a slightly different relative order. Both evaluations ranked a semiautomated method named CAFASP-CONSENSUS, that filed predictions using the CAFASP results of the servers, above any of the individual servers. Although the predictions of the CAFASP servers were available to human CASP predictors before the CASP submission deadline, the CASP assessment identified only 11 human groups that performed better than the best server. Furthermore, about one fourth of the top 30 performing groups corresponded to automated servers. At least half of the top 11 groups corresponded to human groups that also had a server in CAFASP or to human groups that used the CAFASP results to prepare their predictions. In particular, the CAFASP-CONSENSUS group was ranked 7. This shows that the automated predictions of the servers can be very helpful to human predictors. We conclude that as servers continue to improve, they will become increasingly important in any prediction process, especially when dealing with genome-scale prediction tasks. We expect that in the near future, the performance difference between humans and machines will continue to narrow and that fully automated structure prediction will become an effective companion and complement to experimental structural genomics.