RESUMO
Rho-kinase binds to a small GTPase Rho in a GTP-dependent manner and regulates many cytoskeletal events in the cell. The minimum region of bovine Rho-kinase sufficient for Rho-binding was expressed as a fusion protein with glutathione S-transferase. After removal of the glutathione S-transferase, thin plate crystals were obtained. The selenomethionine-substituted protein was introduced and crystallized, as was the native protein. The crystals of the Rho-binding domain of Rho-kinase belong to the space group C2, with unit-cell parameters a = 148.0 (2), b = 26.1 (1), c = 39.6 (1) A, beta = 90.3 (1) degrees. The crystals diffract to a resolution beyond 1.5 A.
Assuntos
Proteínas Serina-Treonina Quinases/química , Animais , Sítios de Ligação , Bovinos , Cristalização , Escherichia coli/genética , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/isolamento & purificação , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rhoRESUMO
Mg(2+) ions are essential for guanosine triphosphatase (GTPase) activity and play key roles in guanine nucleotide binding and preserving the structural integrity of GTP-binding proteins. We determined the crystal structure of a small GTPase RHOA complexed with GDP in the absence of Mg(2+) at 2.0-A resolution. Elimination of a Mg(2+) ion induces significant conformational changes in the switch I region that opens up the nucleotide-binding site. Similar structural changes have been observed in the switch regions of Ha-Ras bound to its guanine nucleotide exchange factor, Sos. This RHOA-GDP structure reveals an important regulatory role for Mg(2+) and suggests that guanine nucleotide exchange factor may utilize this feature of switch I to produce an open conformation in GDP/GTP exchange.
Assuntos
Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Magnésio/análise , Proteína rhoA de Ligação ao GTP/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Processamento de Imagem Assistida por Computador , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Espectrometria de FluorescênciaRESUMO
The effector domain of human protein serine/threonine kinase N (PKN), an effector protein for the small GTP-binding protein Rho, was expressed and purified for protein characterization and crystallization in a complex form with human RhoA. In solution, RhoA binds to the PKN effector domain with 1:2 stoichiometry in a GTP-dependent manner. The obtained complex crystals diffract to 2.2 A resolution.
Assuntos
Proteínas de Ligação ao GTP/química , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/química , Cromatografia em Gel , Cristalização , Guanosina 5'-O-(3-Tiotrifosfato)/química , Humanos , Fragmentos de Peptídeos/química , Ligação Proteica , Proteína Quinase C , Proteínas Recombinantes/química , Difração de Raios XRESUMO
The small G protein Rho has emerged as a key regulator of cellular events involving cytoskeletal reorganization. Here we report the 2.2 A crystal structure of RhoA bound to an effector domain of protein kinase PKN/PRK1. The structure reveals the antiparallel coiled-coil finger (ACC finger) fold of the effector domain that binds to the Rho specificity-determining regions containing switch I, beta strands B2 and B3, and the C-terminal alpha helix A5, predominantly by specific hydrogen bonds. The ACC finger fold is distinct from those for other small G proteins and provides evidence for the diverse ways of effector recognition. Sequence analysis based on the structure suggests that the ACC finger fold is widespread in Rho effector proteins.
