RESUMEN
The citrate synthase (CS) of Escherichia coli is an allosteric hexameric enzyme specifically inhibited by NADH. The crystal structure of wild type (WT) E. coli CS, determined by us previously, has no substrates bound, and part of the active site is in a highly mobile region that is shifted from the position needed for catalysis. The CS of Acetobacter aceti has a similar structure, but has been successfully crystallized with bound substrates: both oxaloacetic acid (OAA) and an analog of acetyl coenzyme A (AcCoA). We engineered a variant of E. coli CS wherein five amino acids in the mobile region have been replaced by those in the A. aceti sequence. The purified enzyme shows unusual kinetics with a low affinity for both substrates. Although the crystal structure without ligands is very similar to that of the WT enzyme (except in the mutated region), complexes are formed with both substrates and the allosteric inhibitor NADH. The complex with OAA in the active site identifies a novel OAA-binding residue, Arg306, which has no functional counterpart in other known CS-OAA complexes. This structure may represent an intermediate in a multi-step substrate binding process where Arg306 changes roles from OAA binding to AcCoA binding. The second complex has the substrate analog, S-carboxymethyl-coenzyme A, in the allosteric NADH-binding site and the AcCoA site is not formed. Additional CS variants unable to bind adenylates at the allosteric site show that this second complex is not a factor in positive allosteric activation of AcCoA binding.
Asunto(s)
Acetobacter/enzimología , Acetilcoenzima A/química , Citrato (si)-Sintasa/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , NADP/química , Acetobacter/genética , Acetilcoenzima A/genética , Acetilcoenzima A/metabolismo , Regulación Alostérica , Animales , Dominio Catalítico , Citrato (si)-Sintasa/genética , Citrato (si)-Sintasa/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , NADP/genética , NADP/metabolismo , Unión Proteica , PorcinosRESUMEN
The recent structure of human replication protein A (RPA) bound to residues 38-58 of tumor suppressor p53 exemplifies several important features of protein-protein interactions involved in transcription and DNA repair. First, the N-terminal transcriptional activation domain (TAD) of p53 is multifunctional and dynamic, showing multiple interactions with partner proteins some of which are modulated by phosphorylation. Second, the binding of partner proteins is coupled with a disorder-to-order transition common to many other transcriptional activation domains. Third, the molecular features of p53 residues 47-58 imitate those of single stranded DNA in their interaction with the oligonucleotide oliogsaccharide-binding (OB) fold of the N-terminal domain of RPA70. This regulated association is implicated in transmitting the DNA damage signal to the p53 pathway of stress response. Here we review the recently reported crystal structure of the p53/RPA70N complex and the mechanism by which ssDNA can provide positive feedback to dissociate p53/RPA complexes. The binding mode and regulatory mechanisms of the p53/RPA70N interaction may represent a general paradigm for regulation of the OB folds involved in DNA repair and metabolism.
Asunto(s)
Activación Transcripcional , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , ADN/metabolismo , Humanos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Proteína de Replicación A/química , Proteína de Replicación A/metabolismoRESUMEN
One of many protein-protein interactions modulated upon DNA damage is that of the single-stranded DNA-binding protein, replication protein A (RPA), with the p53 tumor suppressor. Here we report the crystal structure of RPA residues 1-120 (RPA70N) bound to the N-terminal transactivation domain of p53 (residues 37-57; p53N) and, by using NMR spectroscopy, characterize two mechanisms by which the RPA/p53 interaction can be modulated. RPA70N forms an oligonucleotide/oligosaccharide-binding fold, similar to that previously observed for the ssDNA-binding domains of RPA. In contrast, the N-terminal p53 transactivation domain is largely disordered in solution, but residues 37-57 fold into two amphipathic helices, H1 and H2, upon binding with RPA70N. The H2 helix of p53 structurally mimics the binding of ssDNA to the oligonucleotide/oligosaccharide-binding fold. NMR experiments confirmed that both ssDNA and an acidic peptide mimicking a phosphorylated form of RPA32N can independently compete the acidic p53N out of the binding site. Taken together, our data suggest a mechanism for DNA damage signaling that can explain a threshold response to DNA damage.
Asunto(s)
ADN de Cadena Simple/química , Proteína de Replicación A/química , Activación Transcripcional , Proteína p53 Supresora de Tumor/química , Secuencia de Aminoácidos , Sitios de Unión , Unión Competitiva , Daño del ADN , Humanos , Datos de Secuencia Molecular , Fosforilación , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteína de Replicación A/metabolismoRESUMEN
The BRCA1 tumor suppressor gene encodes an 1863 amino acid gene product that is implicated in many cellular pathways including transcription, cell-cycle checkpoint control, apoptosis and DNA repair. Much attention has been focused on the structural and biochemical characterization of the N-terminal RING and tandem C-terminal BRCT domains of BRCA1. Here we used NMR spectroscopy in conjunction with CD spectroscopy and limited proteolysis to investigate the biophysical properties of the approximately 1500 residue central region of BRCA1. Our results show that although there are a few small, mildly protease-resistant regions, the majority of the BRCA1 central region lacks any pre-existing independently folded globular domains. Electrophoretic mobility shift assay and intrinsic tryptophan fluorescence experiments also demonstrate that, although intrinsically disordered, polypeptides from the central region are able to mediate interactions with DNA and p53 with affinities in the low micromolar range. This supports a model in which the central region may act as a long flexible scaffold for intermolecular interactions, thereby helping to integrate multiple signals in the DNA damage response pathway.
Asunto(s)
Proteína BRCA1/química , ADN/química , Apoptosis , Proteína BRCA1/metabolismo , Fenómenos Biofísicos , Biofisica , Neoplasias de la Mama/metabolismo , Ciclo Celular , Dicroismo Circular , Reparación del ADN , Relación Dosis-Respuesta a Droga , Humanos , Espectroscopía de Resonancia Magnética , Modelos Genéticos , Mutación Missense , Reacción en Cadena de la Polimerasa , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Transcripción Genética , Triptófano/química , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Study of the hexameric and allosterically regulated citrate synthases (type II CS) provides a rare opportunity to gain not only an understanding of a novel allosteric mechanism but also insight into how such properties can evolve from an unregulated structural platform (the dimeric type I CS). To address both of these issues, we have determined the structure of the complex of NADH (a negative allosteric effector) with the F383A variant of type II Escherichia coli CS. This variant was chosen because its kinetics indicate it is primarily in the T or inactive allosteric conformation, the state that strongly binds to NADH. Our structural analyses show that the six NADH binding sites in the hexameric CS complex are located at the interfaces between dimer units such that most of each site is formed by one subunit, but a number of key residues are drawn from the adjacent dimer. This arrangement of interactions serves to explain why NADH allosteric regulation is a feature only of hexameric type II CS. Surprisingly, in both the wild-type enzyme and the NADH complex, the two subunits of each dimer within the hexameric conformation are similar but not identical in structure, and therefore, while the general characteristics of NADH binding interactions are similar in each subunit, the details of these are somewhat different between subunits. Detailed examination of the observed NADH binding sites indicates that both direct charged interactions and the overall cationic nature of the sites are likely responsible for the ability of these sites to discriminate between NADH and NAD(+). A particularly novel characteristic of the complex is the horseshoe conformation assumed by NADH, which is strikingly different from the extended conformation found in its complexes with most proteins. Sequence homology studies suggest that this approach to binding NADH may arise out of the evolutionary need to add an allosteric regulatory function to the base CS structure. Comparisons of the amino acid sequences of known type II CS enzymes, from different Gram-negative bacteria taxonomic groups, show that the NADH-binding residues identified in our structure are strongly conserved, while hexameric CS molecules that are insensitive to NADH have undergone key changes in the sequence of this part of the protein.
Asunto(s)
Citrato (si)-Sintasa/química , Citrato (si)-Sintasa/metabolismo , NAD/metabolismo , Regulación Alostérica , Sitio Alostérico/genética , Secuencia de Aminoácidos , Citrato (si)-Sintasa/clasificación , Citrato (si)-Sintasa/genética , Cristalografía por Rayos X , Dimerización , Escherichia coli/enzimología , Escherichia coli/genética , Evolución Molecular , Variación Genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Cuaternaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
A four-dimensional (4-D) NMR study of Escherichia coli malate synthase G (MSG), a 723-residue monomeric enzyme (81.4 kDa), is described. Virtually complete backbone (1)HN, (15)N, (13)C, and (13)C(beta) chemical shift assignments of this largely alpha-helical protein are reported. The assignment strategy follows from our previously described approach based on TROSY triple resonance 4-D NMR spectroscopy [Yang, D.; Kay, L. E. J. Am. Chem. Soc. 1999, 121, 2571-2575. Konrat, R; Yang, D; Kay, L. E. J. Biomol. NMR 1999, 15, 309-313] with a number of modifications necessitated by the large size of the protein. A protocol for refolding deuterated MSG in vitro was developed to protonate the amides deeply buried in the protein core. Of interest, during the course of the assignment, an isoaspartyl linkage in the protein sequence was unambiguously identified. Chemical shift assignments of this system are a first step in the study of how the domains of the protein change in response to ligand binding and for characterizing the dynamical properties of the enzyme that are likely important for function.