RESUMEN
Determining structures of protein complexes is crucial for understanding cellular functions. Here, we describe an integrative structure determination approach that relies on in vivo measurements of genetic interactions. We construct phenotypic profiles for point mutations crossed against gene deletions or exposed to environmental perturbations, followed by converting similarities between two profiles into an upper bound on the distance between the mutated residues. We determine the structure of the yeast histone H3-H4 complex based on ~500,000 genetic interactions of 350 mutants. We then apply the method to subunits Rpb1-Rpb2 of yeast RNA polymerase II and subunits RpoB-RpoC of bacterial RNA polymerase. The accuracy is comparable to that based on chemical cross-links; using restraints from both genetic interactions and cross-links further improves model accuracy and precision. The approach provides an efficient means to augment integrative structure determination with in vivo observations.
Asunto(s)
Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Mapas de Interacción de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Histonas/química , Histonas/genética , Mutación , Conformación Proteica , Mapeo de Interacción de Proteínas , Saccharomyces cerevisiae/genéticaRESUMEN
Computational prediction and design of membrane protein-protein interactions facilitate biomedical engineering and biotechnological applications. Due to their antimicrobial activity, human defensins play an important role in the innate immune system. Human defensins are attractive pharmaceutical targets due to their small size, broad activity spectrum, reduced immunogenicity, and resistance to proteolysis. Protein engineering based modification of defensins can improve their pharmaceutical properties. Here we present an approach to computationally probe defensins' oligomerization states in the membrane. First, we develop a novel docking and rescoring algorithm. Then, on the basis of the 3D structure of Sapecin, an insect defensin, and a model of its antimicrobial ion-channel, we optimize the parameters of our empirical scoring function. Finally, we apply our docking program and scoring function to the hBD-2 (human ß-defensin-2) molecule and obtain structures of four possible oligomers. These results can be used in higher level simulations.