RESUMEN
Computer simulations using the simplified energy function and simulated tempering dynamics have accurately determined the native structure of the pYVPML, SVLpYTAVQPNE, and SPGEpYVNIEF peptides in the complexes with SH2 domains. Structural and equilibrium aspects of the peptide binding with SH2 domains have been studied by generating temperature-dependent binding free energy landscapes. Once some native peptide-SH2 domain contacts are constrained, the underlying binding free energy profile has the funnel-like shape that leads to a rapid and consistent acquisition of the native structure. The dominant native topology of the peptide-SH2 domain complexes represents an extended peptide conformation with strong specific interactions in the phosphotyrosine pocket and hydrophobic interactions of the peptide residues C-terminal to the pTyr group. The topological features of the peptide-protein interface are primarily determined by the thermodynamically stable phosphotyrosyl group. A diversity of structurally different binding orientations has been observed for the amino-terminal residues to the phosphotyrosine. The dominant native topology for the peptide residues carboxy-terminal to the phosphotyrosine is tolerant to flexibility in this region of the peptide-SH2 domain interface observed in equilibrium simulations. The energy landscape analysis has revealed a broad, entropically favorable topology of the native binding mode for the bound peptides, which is robust to structural perturbations. This could provide an additional positive mechanism underlying tolerance of the SH2 domains to hydrophobic conservative substitutions in the peptide specificity region.
Asunto(s)
Modelos Moleculares , Péptidos/química , Péptidos/metabolismo , Dominios Homologos src , Sitios de Unión , Simulación por Computador , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Método de Montecarlo , Unión Proteica , Temperatura , TermodinámicaRESUMEN
Stochastic search algorithms can be used to perform rapid six-dimensional molecular-replacement searches. A molecular-replacement procedure has been developed that uses an evolutionary algorithm to simultaneously optimize the orientation and position of a search model in a unit cell. Here, the performance of this algorithm and its dependence on search model quality and choice of target function are examined. Although the evolutionary search procedure is capable of finding solutions with search models that represent only a small fraction of the total scattering matter of the target molecule, the efficiency of the search procedure is highly dependent on the quality of the search model. Polyalanine models frequently provide better search efficiency than all-atom models, even in cases where the side-chain positions are known with high accuracy. Although the success of the search procedure is not highly dependent on the statistic used as the target function, the correlation coefficient between observed and calculated structure-factor amplitudes generally results in better search efficiency than does the R factor. An alternative stochastic search procedure, simulated annealing, provides similar overall performance to evolutionary search. Methods of extending the evolutionary search algorithm to include internal optimization, selection and construction of the search model are now beginning to be investigated.
Asunto(s)
Algoritmos , Modelos Moleculares , Cristalografía por Rayos X , Evolución Molecular , Procesos EstocásticosRESUMEN
Common failures in predicting crystal structures of ligand-protein complexes are investigated for three ligand-protein systems by a combined thermodynamic and kinetic analysis of the binding energy landscapes. Misdocked predictions in ligand-protein docking are classified as 'soft' and 'hard' failures. While a soft failure arises when the search algorithm is unable to find the global energy minimum corresponding to the crystal structure, a hard failure results from a flaw of the energy function to qualify the crystal structure as the predicted lowest energy conformation in docking simulations. We find that neither the determination of a single structure with the lowest energy nor finding the most common binding mode is sufficient to predict crystal structures of the complexes, which belong to the category of hard failures. In a proposed hierarchical approach, structural similarity clustering of the conformations, generated from equilibrium simulations with the simplified energy function, is followed by energy refinement with the AMBER force field. This protocol, that involves a hierarchy of energy functions, resolves some common failures in ligand-protein docking and detects crystallographic binding modes that were not found during docking simulations.
Asunto(s)
Proteínas/metabolismo , Cristalografía , Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Cinética , Ligandos , Maltosa/química , Maltosa/metabolismo , Modelos Moleculares , Estructura Molecular , Proteínas/química , TermodinámicaRESUMEN
The thermodynamic and kinetic aspects of molecular recognition for the methotrexate (MTX)-dihydrofolate reductase (DHFR) ligand-protein system are investigated by the binding energy landscape approach. The impact of 'hot' and 'cold' errors in ligand mutations on the thermodynamic stability of the native MTX-DHFR complex is analyzed, and relationships between the molecular recognition mechanism and the degree of ligand optimization are discussed. The nature and relative stability of intermediates and thermodynamic phases on the ligand-protein association pathway are studied, providing new insights into connections between protein folding and molecular recognition mechanisms, and cooperativity of ligand-protein binding. The results of kinetic docking simulations are rationalized based on the thermodynamic properties determined from equilibrium simulations and the shape of the underlying binding energy landscape. We show how evolutionary ligand selection for a receptor active site can produce well-optimized ligand-protein systems such as MTX-DHFR complex with the thermodynamically stable native structure and a direct transition mechanism of binding from unbound conformations to the unique native structure.
Asunto(s)
Simulación por Computador , Modelos Moleculares , Unión Proteica , Animales , Sitios de Unión , Evolución Molecular , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/metabolismo , Antagonistas del Ácido Fólico/farmacología , Ligandos , Sustancias Macromoleculares , Metotrexato/química , Metotrexato/metabolismo , Metotrexato/farmacología , Modelos Químicos , Método de Montecarlo , Conformación Proteica , Pliegue de Proteína , Selección Genética , Relación Estructura-Actividad , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/metabolismo , TermodinámicaRESUMEN
The thermodynamics of ligand-protein molecular recognition is investigated by the energy landscape approach for two systems: methotrexate(MTX)--dihydrofolate reductase(DHFR) and biotin-streptavidin. The temperature-dependent binding free energy profile is determined using the weighted histogram analysis method. Two different force fields are employed in this study: a simplified model of ligand-protein interactions and the AMBER force field with a soft core smoothing component, used to soften the repulsive part of the potential. The results of multiple docking simulations are rationalized from the shape of the binding free energy profile that characterizes the thermodynamics of the binding process.
Asunto(s)
Simulación por Computador , Modelos Químicos , Proteínas/química , Proteínas/metabolismo , Programas Informáticos , Biotina/química , Biotina/metabolismo , Cinética , Ligandos , Metotrexato/química , Metotrexato/metabolismo , Método de Montecarlo , Unión Proteica , Estreptavidina/química , Estreptavidina/metabolismo , Temperatura , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/metabolismo , TermodinámicaRESUMEN
A new procedure for molecular replacement is presented in which an efficient six-dimensional search is carried out using an evolutionary optimization algorithm. In this procedure, a population of initially random molecular-replacement solutions is iteratively optimized with respect to the correlation coefficient between observed and calculated structure factors. The sensitivity and reliability of the method is enhanced by uniform sampling of the rotational-search space and the use of continuously variable rotational and translational parameters. The process is several orders of magnitude faster than a systematic six-dimensional search, and comparisons show that it can identify solutions using significantly less accurate or less complete search models than is possible with two existing molecular-replacement methods. A program incorporating the method, EPMR, allows the rapid and highly automated solution of molecular-replacement problems involving single or multiple molecules in the asymmetric unit. EPMR has been used to solve a number of difficult molecular-replacement problems.
Asunto(s)
Evolución Molecular , Conformación Proteica , Algoritmos , AutomatizaciónRESUMEN
Energy landscapes of molecular recognition are explored by performing "semi-rigid" docking of FK-506 and rapamycin with the Fukisawa binding protein (FKBP-12), and flexible docking simulations of the Ro-31-8959 and AG-1284 inhibitors with HIV-1 protease by a genetic algorithm. The requirements of a molecular recognition model to meet thermodynamic and kinetic criteria of ligand-protein docking simultaneously are investigated using a family of simple molecular recognition energy functions. The critical factor that determines the success rate in predicting the structure of ligand-protein complexes is found to be the roughness of the binding energy landscape, in accordance with a minimal frustration principle. The results suggest that further progress in structure prediction of ligand-protein complexes can be achieved by designing molecular recognition energy functions that generate binding landscapes with reduced frustration.
Asunto(s)
Algoritmos , Proteínas Portadoras/química , Proteínas de Unión al ADN/química , Proteasa del VIH/química , VIH-1/enzimología , Proteínas de Choque Térmico/química , Proteínas Portadoras/genética , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Proteasa del VIH/efectos de los fármacos , Proteasa del VIH/genética , Inhibidores de la Proteasa del VIH/farmacología , Proteínas de Choque Térmico/genética , Modelos Genéticos , Proteínas de Unión a TacrolimusRESUMEN
A series of potent nonpeptide inhibitors of the HIV protease have been identified. Using the structure of compound 3 bound to the HIV protease, bis tertiary amide inhibitor 9 was designed and prepared. Compound 9 was found to be about 17 times more potent than 3, and the structure of the protein-ligand complex of 9 revealed the inhibitor binds in an inverted binding mode relative to 3. Examination of the protein-ligand complex of 9 suggested several modifications in the P1 and P1' pockets. Through these modifications it was possible to improve the activity of the inhibitors another 100-fold, highlighting the utility of crystallographic feedback in inhibitor design. These compounds were found to have good antiviral activity in cell culture, were selective for the HIV protease, and were orally available in three animal models.
Asunto(s)
Amidas/síntesis química , Antivirales/síntesis química , Inhibidores de la Proteasa del VIH/síntesis química , VIH-1/efectos de los fármacos , Amidas/farmacología , Animales , Antivirales/farmacología , Línea Celular , Perros , Diseño de Fármacos , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/enzimología , Haplorrinos , Humanos , Ratones , Estructura Molecular , Ratas , Relación Estructura-ActividadRESUMEN
BACKGROUND: An important prerequisite for computational structure-based drug design is prediction of the structures of ligand-protein complexes that have not yet been experimentally determined by X-ray crystallography or NMR. For this task, docking of rigid ligands is inadequate because it assumes knowledge of the conformation of the bound ligand. Docking of flexible ligands would be desirable, but requires one to search an enormous conformational space. We set out to develop a strategy for flexible docking by combining a simple model of ligand-protein interactions for molecular recognition with an evolutionary programming search technique. RESULTS: We have developed an intermolecular energy function that incorporates steric and hydrogen-bonding terms. The parameters in this function were obtained by docking in three different protein systems. The effectiveness of this method was demonstrated by conformationally flexible docking of the inhibitor AG-1343, a potential new drug against AIDS, into HIV-1 protease. For this molecule, which has nine rotatable bonds, the crystal structure was reproduced within 1.5 A root-mean-square deviation 34 times in 100 simulations, each requiring eight minutes on a Silicon Graphics R4400 workstation. The energy function correctly evaluates the crystal structure as the global energy minimum. CONCLUSIONS: We believe that a solution of the docking problem may be achieved by matching a simple model of molecular recognition with an efficient search procedure. The necessary ingredients of a molecular recognition model include only steric and hydrogen-bond interaction terms. Although these terms are not necessarily sufficient to predict binding affinity, they describe ligand-protein interactions faithfully enough to enable a docking program to predict the structure of the bound ligand. This docking strategy thus provides an important tool for the interdisciplinary field of rational drug design.
Asunto(s)
Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/química , Nelfinavir/farmacología , Evolución Biológica , Cristalografía por Rayos X , Evolución Molecular Dirigida , Diseño de Fármacos , Inhibidores de la Proteasa del VIH/química , Humanos , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Nelfinavir/química , Resonancia Magnética Nuclear Biomolecular , Conformación ProteicaRESUMEN
A new computational method for the in situ generation of small molecules within the binding site of a protein is described. The method has been evaluated using two well-studied systems, dihydrofolate reductase and thymidylate synthase. The method has also been used to guide improvements to inhibitors of HIV-1 protease. One such improvement resulted in a compound selected for preclinical studies as an antiviral agent against AIDS.