RESUMO
The mitochondrial import receptor of 70 kDa, Tom70, preferentially recognizes precursors of membrane proteins with internal targeting signals. We report the identification of a stably folded 25 kDa core domain located in the middle portion of Tom70 that contains two of the seven tetratricopeptide repeat motifs of the receptor. The core domain binds non-cleavable and cleavable preproteins carrying internal targeting signals with a specificity indistinguishable from the full-length receptor. Competition studies indicate that both types of preproteins interact with overlapping binding sites of the core domain and that at least one additional interaction site is present in the full-length receptor. We suggest a model of Tom70 function in import of membrane proteins whereby a hydrophobic preprotein concomitantly interacts with several binding sites of the receptor.
Assuntos
Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Precursores de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Ligação Competitiva/efeitos dos fármacos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Peso Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Dobramento de Proteína , Precursores de Proteínas/química , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Deleção de Sequência/genética , Especificidade por Substrato , Termodinâmica , Tripsina/metabolismoRESUMO
Adrenodoxin reductase is a flavoenzyme that shuffles electrons for the biosynthesis of steroids. Its chain topology belongs to the glutathione reductase family of disulfide oxidoreductases, all of which bind FAD at equivalent positions. The three reported structures of adrenodoxin reductase were ligated with reduced and oxidized NADP and have now confirmed this equivalence also for the NADP-binding site. Remarkably, the conformations and relative positions of the prosthetic group FAD and the cofactor NADP have been conserved during protein evolution despite very substantial changes in the polypeptide. The ligated enzymes showed small changes in the domain positions. When compared with the structure of the NADP-free enzyme, these positions correspond to several states of the domain motion during NADP binding. On the basis of the observed structures, we suggest an enzymatic mechanism for the subdivision of the received two-electron package into the two single electrons transferred to the carrier protein adrenodoxin. The data banks contain 10 sequences that are closely related to bovine adrenodoxin reductase. Most of them code for gene products with unknown functions. Within this family, the crucial residues of adrenodoxin reductase are strictly conserved. Moreover, the putative docking site of the carrier is rather well conserved. Five of the family members were assigned names related to ferredoxin:NADP(+) reductase, presumably because adrenodoxin reductase was considered a member of this functionally similar family. Since this is not the case, the data bank entries should be corrected.
Assuntos
Ferredoxina-NADP Redutase/química , NADP/química , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Bovinos , Cristalização , Cristalografia por Raios X , Transporte de Elétrons/genética , Ferredoxina-NADP Redutase/genética , Ferredoxina-NADP Redutase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , NADP/metabolismo , Estrutura Terciária de Proteína/genética , Alinhamento de SequênciaRESUMO
Adrenodoxin reductase is a monomeric 51 kDa flavoenzyme that is involved in the biosynthesis of all steroid hormones. The structure of the native bovine enzyme was determined at 2.8 A resolution, and the structure of the respective recombinant enzyme at 1.7 A resolution. Adrenodoxin reductase receives a two-electron package from NADPH and converts it to two single electrons that are transferred via adrenodoxin to all mitochondrial cytochromes P 450. The structure suggests how the observed flavin semiquinone is stabilized. A striking feature is the asymmetric charge distribution, which most likely controls the approach of the electron carrier adrenodoxin. A model for the interaction is proposed. Adrenodoxin reductase shows clear sequence homology to half a dozen proteins identified in genome analysis projects, but neither sequence nor structural homology to established, functionally related electron transferases. Yet, the structure revealed a relationship to the disulfide oxidoreductases, permitting the assignment of the NADP-binding site.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Ferredoxina-NADP Redutase/química , Mitocôndrias/metabolismo , Esteroides/biossíntese , Adrenodoxina/metabolismo , Animais , Sítios de Ligação , Bovinos , Transporte de Elétrons , Ferredoxina-NADP Redutase/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Glicosilação , Modelos Moleculares , NADP/metabolismo , Conformação Proteica , Dobramento de Proteína , Tiorredoxina Dissulfeto Redutase/químicaRESUMO
Adrenodoxin reductase is an essential component of the mitochondrial monooxygenase systems that are involved in the synthesis of steroid hormones and related compounds. After removing by mutagenesis a secondary ribosome binding site and an mRNA loop formed between the gene and the vector, large amounts of the enzyme could be produced in Escherichia coli by coexpression with the HSP60-chaperone system. The purified protein was homogeneous enough for reproducible crystallization. The crystals diffracted X-rays isotropically beyond 1.7 A resolution permitting a structure analysis.