RESUMO
Human topoisomerase I (top1) is the molecular target of a diverse set of anticancer compounds, including the camptothecins, indolocarbazoles, and indenoisoquinolines. These compounds bind to a transient top1-DNA covalent complex and inhibit the resealing of a single-strand nick that the enzyme creates to relieve superhelical tension in duplex DNA. (Hertzberg, R. P.; et al. Biochem. 1989, 28, 4629-4638. Hsiang, Y. H.; et al. J. Biol. Chem 1985, 260, 14873-14878. Champoux, J. J. Annu. Rev. Biochem. 2001, 70, 369-413. Stewart, L.; et al. Science 1998, 729, 1534-1541.) We report the X-ray crystal structures of the human top1-DNA complex bound with camptothecin and representative members of the indenoisoquinoline and indolocarbazole classes of top1 poisons. The planar nature of all three structurally diverse classes allows them to intercalate between DNA base pairs at the site of single-strand cleavage. All three classes of compounds have a free electron pair near Arg364, a residue that if mutated confers resistance to all three classes of drugs. The common intercalative binding mode is augmented by unexpected chemotype-specific contacts with amino acid residues Asn352 and Glu356, which adopt alternative side-chain conformations to accommodate the bound compounds. These new X-ray structures explain how very different molecules can stabilize top1-DNA covalent complexes and will aid the rational design of completely novel structural classes of anticancer drugs.
Assuntos
Antineoplásicos/química , DNA Topoisomerases Tipo I/química , DNA/química , Inibidores Enzimáticos/química , Sítios de Ligação , Camptotecina/química , Carbazóis/química , Cristalografia por Raios X , DNA/genética , Resistencia a Medicamentos Antineoplásicos , Humanos , Indenos/química , Indóis/química , Substâncias Intercalantes/química , Isoquinolinas/química , Modelos Moleculares , Estrutura Molecular , Mutação , Inibidores da Topoisomerase IRESUMO
The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO(163)) to a 2.5-A resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO(163) has an antiparallel four-helix bundle fold. The neutralizing Fab mainly recognizes the C-D crossover loop containing the species invariant residue Q111. Titration calorimetric experiments show that hTPO(163) interacts with soluble c-Mpl containing the extracellular cytokine receptor homology domains with 1:2 stoichiometry with the binding constants of 3.3 x 10(9) M(-1) and 1.1 x 10(6) M(-1). The presence of the neutralizing Fab did not inhibit binding of hTPO(163) to soluble c-Mpl fragments, but the lower-affinity binding disappeared. Together with prior genetic data, these define the structure-function relationships in TPO and the activation scheme of c-Mpl.
Assuntos
Fragmentos Fab das Imunoglobulinas/imunologia , Trombopoetina/química , Trombopoetina/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Testes de Neutralização , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Trombopoetina , Solubilidade , Relação Estrutura-Atividade , Especificidade por Substrato , Trombopoetina/imunologiaRESUMO
We report the x-ray crystal structure of human topoisomerase I covalently joined to double-stranded DNA and bound to the clinically approved anticancer agent Topotecan. Topotecan mimics a DNA base pair and binds at the site of DNA cleavage by intercalating between the upstream (-1) and downstream (+1) base pairs. Intercalation displaces the downstream DNA, thus preventing religation of the cleaved strand. By specifically binding to the enzyme-substrate complex, Topotecan acts as an uncompetitive inhibitor. The structure can explain several of the known structure-activity relationships of the camptothecin family of anticancer drugs and suggests that there are at least two classes of mutations that can produce a drug-resistant enzyme. The first class includes changes to residues that contribute to direct interactions with the drug, whereas a second class would alter interactions with the DNA and thereby destabilize the drug-binding site.
Assuntos
Antineoplásicos/farmacologia , DNA/química , Inibidores Enzimáticos/farmacologia , Substâncias Intercalantes/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores da Topoisomerase I , Topotecan/farmacologia , Antineoplásicos/química , Cristalografia por Raios X , DNA/metabolismo , DNA Topoisomerases Tipo I/química , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/química , Humanos , Ligação de Hidrogênio , Substâncias Intercalantes/química , Substâncias Macromoleculares , Modelos Moleculares , Estrutura Molecular , Mutação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/fisiologia , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/fisiologia , Relação Estrutura-Atividade , Topotecan/químicaRESUMO
Thrombopoietin (TPO) is a cytokine which primarily stimulates megakaryocytopoiesis and thrombopoiesis. The functional domain of TPO (TPO(163)) consisting of the N-terminal 163 amino acids was prepared and crystallized. Since the crystallization of TPO(163) was unsuccessful using the standard screening methods, a Fab fragment derived from a neutralizing monoclonal antibody was used for crystallization. It was found that the TPO(163)-Fab complex crystallized reproducibly in 0.1 M potassium phosphate buffer pH 6.0 containing 20-25% polyethylene glycol 4000. Thin crystals (0.2 x 0.2 x 0.02 mm) grew in two space groups: P2(1), with unit-cell parameters a = 133.20, b = 46.71, c = 191.47 A, beta = 90.24 degrees, and C2, with unit-cell parameters a = 131.71, b = 46.48, c = 184.63 A, beta = 90.42 degrees. The results of a molecular-replacement analysis indicate that the Fab molecules interact with each other and provide a suitable interface for crystallization.