RESUMEN
Phosphodiesterase catalyzes the hydrolysis of the intracellular second messenger 3',5'-cyclic AMP (cAMP) into the corresponding 5'-nucleotide. Phosphodiesterase 4 (PDE4), the major cAMP-specific PDE in inflammatory and immune cells, is an attractive target for the treatment of asthma and COPD. We have determined crystal structures of the catalytic domain of PDE4B complexed with AMP (2.0 A), 8-Br-AMP (2.13 A) and the potent inhibitor rolipram (2.0 A). All the ligands bind in the same hydrophobic pocket and can interact directly with the active site metal ions. The identity of these metal ions was examined using X-ray anomalous difference data. The structure of the AMP complex confirms the location of the catalytic site and allowed us to speculate about the detailed mechanism of catalysis. The high-resolution structures provided the experimental insight into the nucleotide selectivity of phosphodiesterase. 8-Br-AMP binds in the syn conformation to the enzyme and demonstrates an alternative nucleotide-binding mode. Rolipram occupies much of the AMP-binding site and forms two hydrogen bonds with Gln443 similar to the nucleotides.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/química , Adenosina Trifosfato/análogos & derivados , 3',5'-AMP Cíclico Fosfodiesterasas/genética , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Adenosina Monofosfato/química , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Humanos , Técnicas In Vitro , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Inhibidores de Fosfodiesterasa/química , Estructura Terciaria de Proteína , Rolipram/química , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
Obtaining diffraction-quality crystals has long been a bottleneck in solving the three-dimensional structures of proteins. Often proteins may be stabilized when they are complexed with a substrate, nucleic acid, cofactor or small molecule. These ligands, on the other hand, have the potential to induce significant conformational changes to the protein and ab initio screening may be required to find a new crystal form. This paper presents an overview of strategies in the following areas for obtaining crystals of protein-ligand complexes: (i) co-expression of the protein with the ligands of interest, (ii) use of the ligands during protein purification, (iii) cocrystallization and (iv) soaks.
Asunto(s)
Cristalización , Cristalografía por Rayos X/métodos , Proteínas/química , Animales , Sitios de Unión , Proteínas Portadoras/química , Humanos , Ligandos , Liposomas/química , Conformación Molecular , Mutación , Receptores Androgénicos/química , Receptores de Glucocorticoides/química , Receptores de Mineralocorticoides/química , TemperaturaRESUMEN
Peptide antagonists of the human papillomavirus type 11 (HPV-11) E2-DNA association were identified using a filamentous bacteriophage random peptide library. Synthetic peptides antagonized the E2-DNA interaction, effectively blocked E2-mediated transcriptional activation of a reporter gene in cell culture, and inhibited E1-E2-mediated HPV-11 DNA replication in vitro. These peptides may prove to be useful tools for characterizing E2 function and for exploring the effectiveness of E2-inhibitor-based treatments for HPV-associated diseases.