RESUMO
Human muscarinic receptor M2 is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M2 receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptors compared to M3 receptors leads to subtype selectivity of AF-DX 384.
Assuntos
Antagonistas Muscarínicos/metabolismo , Pirenzepina/análogos & derivados , Receptor Muscarínico M2/química , Receptor Muscarínico M2/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Estabilidade Enzimática , Humanos , Simulação de Dinâmica Molecular , Antagonistas Muscarínicos/química , Mutação , N-Metilescopolamina/química , N-Metilescopolamina/metabolismo , Pirenzepina/química , Pirenzepina/metabolismo , Receptor Muscarínico M2/antagonistas & inibidoresRESUMO
Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OX2R structures in complex with selective antagonists and previously determined OX1R/OX2R structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OX2R. The importance of these residues for binding selectivity to OX2R was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors.
Assuntos
Aminopiridinas/química , Azepinas/química , Antagonistas dos Receptores de Orexina/química , Receptores de Orexina/química , Orexinas/química , Sulfonamidas/química , Triazóis/química , Aminopiridinas/metabolismo , Animais , Azepinas/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia/métodos , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Ligação de Hidrogênio , Cinética , Simulação de Dinâmica Molecular , Antagonistas dos Receptores de Orexina/metabolismo , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Orexinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Sulfonamidas/metabolismo , Síncrotrons , Termodinâmica , Triazóis/metabolismoRESUMO
During homologous recombination, eukaryotic RecA homologue Rad51 assembles into a nucleoprotein filament on single-stranded DNA to catalyse homologous pairing and DNA-strand exchange with a homologous template. Rad51 nucleoprotein filaments are highly dynamic and regulated via the coordinated actions of various accessory proteins including Rad51 mediators. Here, we identify a new Rad51 mediator complex. The PCSS complex, comprising budding yeast Psy3, Csm2, Shu1 and Shu2 proteins, binds to recombination sites and is required for Rad51 assembly and function during meiosis. Within the hetero-tetramer, Psy3-Csm2 constitutes a core sub-complex with DNA-binding activity. In vitro, purified Psy3-Csm2 stabilizes the Rad51-single-stranded DNA complex independently of nucleotide cofactor. The mechanism of Rad51 stabilization is inferred by our high-resolution crystal structure, which reveals Psy3-Csm2 to be a structural mimic of the Rad51-dimer, a fundamental unit of the Rad51-filament. Together, these results reveal a novel molecular mechanism for this class of Rad51-mediators, which includes the human Rad51 paralogues.
Assuntos
Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Primers do DNA , DNA de Cadeia Simples/metabolismo , Meiose , Recombinação Genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The human centromere protein B (CENP-B), a centromeric heterochromatin component, forms a homodimer that specifically binds to a distinct DNA sequence (the CENP-B box), which appears within every other alpha-satellite repeat. Previously, we determined the structure of the human CENP-B DNA-binding domain, CENP-B-(1-129), complexed with the CENP-B box DNA. In the present study, we determined the crystal structure of its dimerization domain (CENP-B-(540-599)), another functional domain of CENP-B, at 1.65-A resolution. CENP-B-(540-599) contains two alpha-helices, which are folded into an antiparallel configuration. The CENP-B-(540-599) dimer formed a symmetrical, antiparallel, four-helix bundle structure with a large hydrophobic patch in which 23 residues of one monomer form van der Waals contacts with the other monomer. In the CENP-B-(540-599) dimer, the N-terminal ends of CENP-B-(540-599) are oriented on opposite sides of the dimer. This CENP-B dimer configuration may be suitable for capturing two distant CENP-B boxes during centromeric heterochromatin formation.