RESUMEN
The great power of protein crystallography to reveal biological structure is often limited by the tremendous effort required to produce suitable crystals. A hybrid crystal growth predictive model is presented that combines both experimental and sequence-derived data from target proteins, including novel variables derived from physico-chemical characterization such as R(30), the ratio between a protein's DSF intensity at 30°C and at T(m). This hybrid model is shown to be more powerful than sequence-based prediction alone - and more likely to be useful for prioritizing and directing the efforts of structural genomics and individual structural biology laboratories.
Asunto(s)
Modelos Moleculares , Proteínas/química , Cristalización , Cristalografía por Rayos X , Interpretación Estadística de Datos , Análisis de Secuencia de ProteínaRESUMEN
Purine nucleoside phosphorylases (PNPs) and uridine phosphorylases (UPs) are closely related enzymes involved in purine and pyrimidine salvage, respectively, which catalyze the removal of the ribosyl moiety from nucleosides so that the nucleotide base may be recycled. Parasitic protozoa generally are incapable of de novo purine biosynthesis; hence, the purine salvage pathway is of potential therapeutic interest. Information about pyrimidine biosynthesis in these organisms is much more limited. Though all seem to carry at least a subset of enzymes from each pathway, the dependency on de novo pyrimidine synthesis versus salvage varies from organism to organism and even from one growth stage to another. We have structurally and biochemically characterized a putative nucleoside phosphorylase (NP) from the pathogenic protozoan Trypanosoma brucei and find that it is a homodimeric UP. This is the first characterization of a UP from a trypanosomal source despite this activity being observed decades ago. Although this gene was broadly annotated as a putative NP, it was widely inferred to be a purine nucleoside phosphorylase. Our characterization of this trypanosomal enzyme shows that it is possible to distinguish between PNP and UP activity at the sequence level based on the absence or presence of a characteristic UP-specificity insert. We suggest that this recognizable feature may aid in proper annotation of the substrate specificity of enzymes in the NP family.
Asunto(s)
Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/enzimología , Uridina Fosforilasa/química , Uridina Fosforilasa/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Cartilla de ADN/genética , ADN Protozoario/genética , Genes Protozoarios , Metales/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Trypanosoma brucei brucei/genética , Uridina Fosforilasa/antagonistas & inhibidores , Uridina Fosforilasa/genéticaRESUMEN
The 1.8 A resolution de novo structure of nucleoside 2-deoxyribosyltransferase (EC 2.4.2.6) from Trypanosoma brucei (TbNDRT) has been determined by SADa phasing in an unliganded state and several ligand-bound states. This enzyme is important in the salvage pathway of nucleoside recycling. To identify novel lead compounds, we exploited "fragment cocktail soaks". Out of 304 compounds tried in 31 cocktails, four compounds could be identified crystallographically in the active site. In addition, we demonstrated that very short soaks of approximately 10 s are sufficient even for rather hydrophobic ligands to bind in the active site groove, which is promising for the application of similar soaking experiments to less robust crystals of other proteins.