RESUMO
We present here the x-ray structures of the progesterone receptor (PR) in complex with two mixed profile PR modulators whose functional activity results from two differing molecular mechanisms. The structure of Asoprisnil bound to the agonist state of PR demonstrates the contribution of the ligand to increasing stability of the agonist conformation of helix-12 via a specific hydrogen-bond network including Glu(723). This interaction is absent when the full antagonist, RU486, binds to PR. Combined with a previously reported structure of Asoprisnil bound to the antagonist state of the receptor, this structure extends our understanding of the complex molecular interactions underlying the mixed agonist/antagonist profile of the compound. In addition, we present the structure of PR in its agonist conformation bound to the mixed profile compound Org3H whose reduced antagonistic activity and increased agonistic activity compared with reference antagonists is due to an induced fit around Trp(755), resulting in a decreased steric clash with Met(909) but inducing a new internal clash with Val(912) in helix-12. This structure also explains the previously published observation that 16α attachments to RU486 analogs induce mixed profiles by altering the binding of 11ß substituents. Together these structures further our understanding of the steric and electrostatic factors that contribute to the function of steroid receptor modulators, providing valuable insight for future compound design.
Assuntos
Estrenos/química , Mifepristona/química , Oximas/química , Receptores de Progesterona/agonistas , Receptores de Progesterona/química , Cristalografia por Raios X , Humanos , Ligantes , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The progesterone receptor is able to bind to a large number and variety of ligands that elicit a broad range of transcriptional responses ranging from full agonism to full antagonism and numerous mixed profiles inbetween. We describe here two new progesterone receptor ligand binding domain x-ray structures bound to compounds from a structurally related but functionally divergent series, which show different binding modes corresponding to their agonistic or antagonistic nature. In addition, we present a third progesterone receptor ligand binding domain dimer bound to an agonist in monomer A and an antagonist in monomer B, which display binding modes in agreement with the earlier observation that agonists and antagonists from this series adopt different binding modes.
Assuntos
Receptores de Progesterona/agonistas , Receptores de Progesterona/antagonistas & inibidores , Receptores de Progesterona/metabolismo , Animais , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X/métodos , Dimerização , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Ligantes , Mifepristona/química , Modelos Moleculares , Conformação Molecular , Noretindrona/química , Progesterona/química , Ligação Proteica , Conformação ProteicaRESUMO
The mammalian complement system is a phylogenetically ancient cascade system that has a major role in innate and adaptive immunity. Activation of component C3 (1,641 residues) is central to the three complement pathways and results in inflammation and elimination of self and non-self targets. Here we present crystal structures of native C3 and its final major proteolytic fragment C3c. The structures reveal thirteen domains, nine of which were unpredicted, and suggest that the proteins of the alpha2-macroglobulin family evolved from a core of eight homologous domains. A double mechanism prevents hydrolysis of the thioester group, essential for covalent attachment of activated C3 to target surfaces. Marked conformational changes in the alpha-chain, including movement of a critical interaction site through a ring formed by the domains of the beta-chain, indicate an unprecedented, conformation-dependent mechanism of activation, regulation and biological function of C3.
Assuntos
Complemento C3/química , Complemento C3/imunologia , Evolução Molecular , Ativação do Complemento , Complemento C3/metabolismo , Convertases de Complemento C3-C5/metabolismo , Complemento C3c/química , Complemento C3c/metabolismo , Cristalografia por Raios X , Humanos , Modelos Biológicos , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Targeting protein kinases in cancer therapy with irreversible small-molecule inhibitors is moving to the forefront of kinase-inhibitor research and is thought to be an effective means of overcoming mutation-associated drug resistance in epidermal growth factor receptor kinase (EGFR). We generated a detection technique that allows direct measurements of covalent bond formation without relying on kinase activity, thereby allowing the straightforward investigation of the influence of steric clashes on covalent inhibitors in different resistant kinase mutants. The obtained results are discussed together with structural biology and biochemical studies of catalytic activity in both wild-type and gatekeeper mutated kinase variants to draw conclusions about the impact of steric hindrance and increased catalytic activity in drug-resistant kinase variants.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Espectrometria de Fluorescência/métodos , Animais , Galinhas , Cristalografia por Raios X , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Mutação , Neoplasias/tratamento farmacológico , Proteínas Quinases/química , Proteínas Quinases/genética , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/química , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
C2a provides the catalytic center to the convertase complexes of the classical and lectin-binding pathways of complement activation. We determined two crystal structures of full-length C2a, with and without a pseudo ligand bound. Both structures reveal a near-active conformation of the catalytic center of the serine protease domains, while the von Willebrand factor A-type domains display an intermediate activation state of helix alpha7 with an open, activated metal-ion-dependent adhesion site. The open adhesion site likely serves to enhance the affinity for the ligand C4b, similar to "inside-out" signaling in integrins. Surprisingly, the N-terminal residues of C2a are buried in a crevice near helix alpha7, indicative of a structural switch between C2 and C2a. Extended loops on the protease domain possibly envelop the protruding anaphylatoxin domain of the substrate C3. Together with a putative substrate-induced completion of the oxyanion hole, this may contribute to the high substrate specificity of the convertases.
Assuntos
Complemento C2a/química , Modelos Moleculares , Aminoácidos/química , Aminoácidos/genética , Domínio Catalítico , Ativação do Complemento , Complemento C2a/genética , Humanos , Ligantes , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por SubstratoRESUMO
Here we describe the 1.95 A structure of the clinically used antiprogestin RU486 (mifepristone) in complex with the progesterone receptor (PR). The structure was obtained by taking a crystal of the PR ligand binding domain containing the agonist norethindrone and soaking it in a solution containing the antagonist RU486 for extended times. Clear ligand exchange could be observed in one copy of the PR ligand binding domain dimer in the crystal. RU486 binds while PR is in an agonistic conformation without displacing helix 12. Although this is probably because of the constraints of the crystal lattice, it demonstrates that helix 12 displacement is not a prerequisite for RU486 binding. Interestingly, B-factor analysis clearly shows that helix 12 becomes more flexible after RU486 binding, suggesting that RU486, being a model antagonist, does not induce one fixed conformation of helix 12 but changes its positional equilibrium. This conclusion is confirmed by comparing the structures of RU486 bound to PR and RU486 bound to the glucocorticoid receptor.
Assuntos
Mifepristona/química , Receptores de Progesterona/química , Sítios de Ligação , Cristalografia por Raios X , Antagonistas de Hormônios/química , Antagonistas de Hormônios/farmacologia , Humanos , Mifepristona/farmacologia , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/antagonistas & inibidores , Receptores de Glucocorticoides/química , Receptores de Progesterona/agonistas , Receptores de Progesterona/antagonistas & inibidoresRESUMO
Re-evaluation of the crystallographic data of the molybdenum-containing E. coli formate dehydrogenase H (Boyington et al. Science 275:1305-1308, 1997), reported in two redox states, reveals important structural differences for the formate-reduced form, with large implications for the reaction mechanism proposed in that work. We have re-refined the reduced structure with revised protocols and found substantial rearrangement in some parts of it. The original model is essentially correct but an important loop close to the molybdenum active site was mistraced, and, therefore, catalytic relevant residues were located in wrong positions. In particular selenocysteine-140, a ligand of molybdenum in the original work, and essential for catalysis, is no longer bound to the metal after reduction of the enzyme with formate. These results are incompatible with the originally proposed reaction mechanism. On the basis of our new interpretation, we have revised and proposed a new reaction mechanism, which reconciles the new X-ray model with previous biochemical and extended X-ray absorption fine structure data.