RESUMEN
A protocol was developed for the computational determination of the contribution of interfacial amino acid residues to the free energy of protein-protein binding. Thermodynamic integration, based on molecular dynamics simulation in CHARMM, was used to determine the free energy associated with single point mutations to glycine in a protein-protein interface. The hot spot amino acids found in this way were then correlated to structural similarity scores detected by the ProBiS algorithm for local structural alignment. We find that amino acids with high structural similarity scores contribute on average -3.19 kcal/mol to the free energy of protein-protein binding and are thus correlated with hot spot residues, while residues with low similarity scores contribute on average only -0.43 kcal/mol. This suggests that the local structural alignment method provides a good approximation of the contribution of a residue to the free energy of binding and is particularly useful for detection of hot spots in proteins with known structures but undetermined protein-protein complexes.
Asunto(s)
Algoritmos , Aminoácidos/química , Proteínas/química , Programas Informáticos , Sustitución de Aminoácidos , Sitios de Unión , Bases de Datos de Proteínas , Entropía , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Homología Estructural de Proteína , TermodinámicaRESUMEN
D-Alanine:D-alanine ligase (Ddl) is an essential ATP-dependent bacterial enzyme involved in peptidoglycan biosynthesis. Discovery of Ddl inhibitors not competitive with ATP has proven to be difficult because the Ddl bimolecular d-alanine binding pocket is very restricted, as is accessibility to the active site for larger molecules in the catalytically active closed conformation of Ddl. A molecular dynamics study of the opening and closing of the Ddl lid loop informs future structure-based design efforts that allow for the flexibility of Ddl. A virtual screen on generated enzyme conformations yielded some hit inhibitors whose bioactivity was determined.
Asunto(s)
Proteínas Bacterianas/química , Simulación de Dinámica Molecular , Péptido Sintasas/química , Proteínas Bacterianas/antagonistas & inhibidores , Benzotiazoles/síntesis química , Benzotiazoles/química , Pruebas de Enzimas , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/química , Isoxazoles/síntesis química , Isoxazoles/química , Péptido Sintasas/antagonistas & inhibidores , Unión Proteica , Conformación Proteica , Pirazinas/síntesis química , Pirazinas/química , Pirimidinas/síntesis química , Pirimidinas/química , Estereoisomerismo , Relación Estructura-Actividad , Thermus/enzimologíaRESUMEN
D-Alanine:D-alanine ligase (Ddl), an intracellular bacterial enzyme essential for cell wall biosynthesis, is an attractive target for development of novel antimicrobial drugs. This study focused on an extensive evaluation of two families of Ddl inhibitors encountered in our previous research. New members of both families were obtained through similarity search and synthesis. Ellipticines and 9-acridinylamines were both found to possess inhibitory activity against Ddl from Escherichia coli and antimicrobial activity against E. coli and Staphylococcus aureus. Ellipticines with a quaternary methylpyridinium moiety were the most potent among all studied compounds, with MIC values as low as 2 mg/L in strains with intact efflux mechanisms. Antimicrobial activity of the studied compounds was connected to membrane damage, making their development as antibacterial drug candidates unlikely unless analogues devoid of this nonspecific effect can be discovered.
Asunto(s)
Aminas/química , Antiinfecciosos/química , Elipticinas/química , Inhibidores Enzimáticos/química , Péptido Sintasas/antagonistas & inhibidores , Aminas/síntesis química , Aminas/farmacología , Antiinfecciosos/síntesis química , Antiinfecciosos/farmacología , Técnicas Químicas Combinatorias , Elipticinas/síntesis química , Elipticinas/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Escherichia coli/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Péptido Sintasas/metabolismo , Staphylococcus aureus/efectos de los fármacosRESUMEN
Generalization of an earlier algorithm has led to the development of new local structural alignment algorithms for prediction of protein-protein binding sites. The algorithms use maximum cliques on protein graphs to define structurally similar protein regions. The search for structural neighbors in the new algorithms has been extended to all the proteins in the PDB and the query protein is compared to more than 60,000 proteins or over 300,000 single-chain structures. The resulting structural similarities are combined and used to predict the protein binding sites. This study shows that the location of protein binding sites can be predicted by comparing only local structural similarities irrespective of general protein folds.
Asunto(s)
Algoritmos , Proteínas/química , Proteínas/metabolismo , Alineación de Secuencia/métodos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bases de Datos de Proteínas , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
The terminal dipeptide, D-Ala-D-Ala, of the peptidoglycan precursor UDPMurNAc-pentapetide is a crucial building block involved in peptidoglycan cross-linking. It is synthesized in the bacterial cytoplasm by the enzyme d-alanine:d-alanine ligase (Ddl). Structure-based virtual screening of the NCI diversity set of almost 2000 compounds was performed with a DdlB isoform from Escherichia coli using the computational tool AutoDock 4.0. The 130 best-ranked compounds from this screen were tested in an in vitro assay for their inhibition of E. coli DdlB. Three compounds were identified that inhibit the enzyme with K(i) values in micromolar range. Two of these also have promising antibacterial activities against Gram-positive and Gram-negative bacteria.
