RESUMO
The Kir3.1 K(+) channel participates in heart rate control and neuronal excitability through G-protein and lipid signaling pathways. Expression in Escherichia coli has been achieved by replacing three fourths of the transmembrane pore with the pore of a prokaryotic Kir channel, leaving the cytoplasmic pore and membrane interfacial regions of Kir3.1 origin. Two structures were determined at 2.2 A. The selectivity filter is identical to the Streptomyces lividans K(+) channel within error of measurement (r.m.s.d.<0.2 A), suggesting that K(+) selectivity requires extreme conservation of three-dimensional structure. Multiple K(+) ions reside within the pore and help to explain voltage-dependent Mg(2+) and polyamine blockade and strong rectification. Two constrictions, at the inner helix bundle and at the apex of the cytoplasmic pore, may function as gates: in one structure the apex is open and in the other, it is closed. Gating of the apex is mediated by rigid-body movements of the cytoplasmic pore subunits. Phosphatidylinositol 4,5-biphosphate-interacting residues suggest a possible mechanism by which the signaling lipid regulates the cytoplasmic pore.
Assuntos
Proteínas de Bactérias/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Modelos Moleculares , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Sítios de Ligação , Burkholderia/metabolismo , Cristalografia por Raios X , Escherichia coli/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/biossíntese , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Ativação do Canal Iônico , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
The three-dimensional structure of the N-terminal SH3 domain (residues 583-660) of murine Vav, which contains a tetra-proline sequence (Pro 607-Pro 610), was determined by NMR. The solution structure of the SH3 domain shows a typical SH3 fold, but it exists in two conformations due to cis-trans isomerization at the Gly614-Pro615 bond. The NMR structure of the P615G mutant, where Pro615 is replaced by glycine, reveals that the tetra-proline region is inserted into the RT-loop and binds to its own SH3 structure. The C-terminal SH3 domain of Grb2 specifically binds to the trans form of the N-terminal SH3 domain of Vav. The surface of Vav N-terminal SH3 which binds to Grb2 C-terminal SH3 was elucidated by chemical shift mapping experiments using NMR. The surface does not involve the tetra-proline region but involves the region comprising the n-src loop, the N-terminal and the C-terminal regions. This surface is located opposite to the tetra-proline containing region, consistent with that of our previous mutagenesis studies.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas de Ciclo Celular , Proteínas/química , Proteínas Proto-Oncogênicas/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteína Adaptadora GRB2 , Isomerismo , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Conformação Proteica , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-vav , Soluções , Domínios de Homologia de srcRESUMO
Inward rectifier K(+) channels govern the resting membrane voltage in many cells. Regulation of these ion channels via G protein-coupled receptor signaling underlies the control of heart rate and the actions of neurotransmitters in the central nervous system. We have determined the protein structure formed by the intracellular N- and C termini of the G protein-gated inward rectifier K(+) channel GIRK1 at 1.8 A resolution. A cytoplasmic pore, conserved among inward rectifier K(+) channels, extends the ion pathway to 60 A, nearly twice the length of a canonical transmembrane K(+) channel. The cytoplasmic pore is lined by acidic and hydrophobic amino acids, creating a favorable environment for polyamines, which block the pore. These results explain in structural and chemical terms the basis of inward rectification, and they also have implications for G protein regulation of GIRK channels.