RESUMEN
Human factor D (FD) is a self-inhibited thrombin-like serine proteinase that is critical for amplification of the complement immune response. FD is activated by its substrate through interactions outside the active site. The substrate-binding, or `exosite', region displays a well defined and rigid conformation in FD. In contrast, remarkable flexibility is observed in thrombin and related proteinases, in which Na(+) and ligand binding is implied in allosteric regulation of enzymatic activity through protein dynamics. Here, ensemble refinement (ER) of FD and thrombin crystal structures is used to evaluate structure and dynamics simultaneously. A comparison with previously published NMR data for thrombin supports the ER analysis. The R202A FD variant has enhanced activity towards artificial peptides and simultaneously displays active and inactive conformations of the active site. ER revealed pronounced disorder in the exosite loops for this FD variant, reminiscent of thrombin in the absence of the stabilizing Na(+) ion. These data indicate that FD exhibits conformational dynamics like thrombin, but unlike in thrombin a mechanism has evolved in FD that locks the unbound native state into an ordered inactive conformation via the self-inhibitory loop. Thus, ensemble refinement of X-ray crystal structures may represent an approach alternative to spectroscopy to explore protein dynamics in atomic detail.
Asunto(s)
Simulación de Dinámica Molecular , Dominio Catalítico/genética , Factor D del Complemento/antagonistas & inhibidores , Factor D del Complemento/química , Factor D del Complemento/genética , Cristalografía por Rayos X , Células HEK293 , Humanos , Mutación , Conformación Proteica , Proteolisis , Relación Estructura-Actividad , Especificidad por Sustrato/genética , Trombina/química , Trombina/genéticaRESUMEN
Single-structure models derived from X-ray data do not adequately account for the inherent, functionally important dynamics of protein molecules. We generated ensembles of structures by time-averaged refinement, where local molecular vibrations were sampled by molecular-dynamics (MD) simulation whilst global disorder was partitioned into an underlying overall translation-libration-screw (TLS) model. Modeling of 20 protein datasets at 1.1-3.1 Å resolution reduced cross-validated R(free) values by 0.3-4.9%, indicating that ensemble models fit the X-ray data better than single structures. The ensembles revealed that, while most proteins display a well-ordered core, some proteins exhibit a 'molten core' likely supporting functionally important dynamics in ligand binding, enzyme activity and protomer assembly. Order-disorder changes in HIV protease indicate a mechanism of entropy compensation for ordering the catalytic residues upon ligand binding by disordering specific core residues. Thus, ensemble refinement extracts dynamical details from the X-ray data that allow a more comprehensive understanding of structure-dynamics-function relationships.DOI:http://dx.doi.org/10.7554/eLife.00311.001.
Asunto(s)
Modelos Estadísticos , Simulación de Dinámica Molecular , Proteínas/química , Programas Informáticos , Animales , Bacterias/química , Cristalografía por Rayos X , Entropía , Humanos , Ligandos , Plantas/química , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Virus/químicaRESUMEN
Here we present a suite of pulse sequences for the measurement of (15)N T(1), T(1rho) and NOE data that combine traditional TROSY-based pulse sequences with band-selective Hadamard frequency encoding. The additive nature of the Hadamard matrix produces much reduced resonance overlap without the need for an increase in the dimensionality of the experiment or a significant decrease in the signal to noise ratio. We validate the accuracy of these sequences in application to ubiquitin and demonstrate their utility for relaxation measurements in Escherichia coli Class II fructose 1,6-bisphosphate aldolase (FBP-aldolase), a 358 residue 78 kDa dimeric enzyme.