RESUMEN
High-resolution NMR spectroscopy enabled us to characterize allosteric transitions between various functional states of the dimeric Escherichia coli Lac repressor. In the absence of ligands, the dimer exists in a dynamic equilibrium between DNA-bound and inducer-bound conformations. Binding of either effector shifts this equilibrium toward either bound state. Analysis of the ternary complex between repressor, operator DNA, and inducer shows how adding the inducer results in allosteric changes that disrupt the interdomain contacts between the inducer binding and DNA binding domains and how this in turn leads to destabilization of the hinge helices and release of the Lac repressor from the operator. Based on our data, the allosteric mechanism of the induction process is in full agreement with the well-known Monod-Wyman-Changeux model.
Asunto(s)
Proteínas de Escherichia coli , Represoras Lac/genética , Represoras Lac/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulación Alostérica/genética , Escherichia coli/metabolismo , ADN/metabolismo , Estructura Secundaria de Proteína , Operón Lac/genéticaRESUMEN
The nucleotide excision repair protein complex ERCC1-XPF is required for incision of DNA upstream of DNA damage. Functional studies have provided insights into the binding of ERCC1-XPF to various DNA substrates. However, because no structure for the ERCC1-XPF-DNA complex has been determined, the mechanism of substrate recognition remains elusive. Here we biochemically characterize the substrate preferences of the helix-hairpin-helix (HhH) domains of XPF and ERCC-XPF and show that the binding to single-stranded DNA (ssDNA)/dsDNA junctions is dependent on joint binding to the DNA binding domain of ERCC1 and XPF. We reveal that the homodimeric XPF is able to bind various ssDNA sequences but with a clear preference for guanine-containing substrates. NMR titration experiments and in vitro DNA binding assays also show that, within the heterodimeric ERCC1-XPF complex, XPF specifically recognizes ssDNA. On the other hand, the HhH domain of ERCC1 preferentially binds dsDNA through the hairpin region. The two separate non-overlapping DNA binding domains in the ERCC1-XPF heterodimer jointly bind to an ssDNA/dsDNA substrate and, thereby, at least partially dictate the incision position during damage removal. Based on structural models, NMR titrations, DNA-binding studies, site-directed mutagenesis, charge distribution, and sequence conservation, we propose that the HhH domain of ERCC1 binds to dsDNA upstream of the damage, and XPF binds to the non-damaged strand within a repair bubble.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Sitios de Unión , Proteínas de Unión al ADN/química , Dimerización , Secuencias Hélice-Asa-Hélice , Humanos , Unión Proteica , Especificidad por SustratoRESUMEN
The ERCC1-XPF heterodimer, a structure-specific DNA endonuclease, is best known for its function in the nucleotide excision repair (NER) pathway. The ERCC1 point mutation F231L, located at the hydrophobic interaction interface of ERCC1 (excision repair cross-complementation group 1) and XPF (xeroderma pigmentosum complementation group F), leads to severe NER pathway deficiencies. Here, we analyze biophysical properties and report the NMR structure of the complex of the C-terminal tandem helix-hairpin-helix domains of ERCC1-XPF that contains this mutation. The structures of wild type and the F231L mutant are very similar. The F231L mutation results in only a small disturbance of the ERCC1-XPF interface, where, in contrast to Phe(231), Leu(231) lacks interactions stabilizing the ERCC1-XPF complex. One of the two anchor points is severely distorted, and this results in a more dynamic complex, causing reduced stability and an increased dissociation rate of the mutant complex as compared with wild type. These data provide a biophysical explanation for the severe NER deficiencies caused by this mutation.
Asunto(s)
Síndrome de Cockayne/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Mutación Puntual , Secuencia de Aminoácidos , Proteínas de Unión al ADN/química , Dimerización , Endonucleasas/química , Humanos , Modelos Químicos , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA. Recognition of altered DNA, including bulky adducts, pyrimidine dimers and interstrand crosslinks (ICL), partially depends on proteins containing helix-hairpin-helix (HhH) domains. To understand how ICL is specifically recognized by the Fanconi anemia proteins FANCM and FAAP24, we determined the structure of the HhH domain of FAAP24. Although it resembles other HhH domains, the FAAP24 domain contains a canonical hairpin motif followed by distorted motif. The HhH domain can bind various DNA substrates; using nuclear magnetic resonance titration experiments, we demonstrate that the canonical HhH motif is required for double-stranded DNA (dsDNA) binding, whereas the unstructured N-terminus can interact with single-stranded DNA. Both DNA binding surfaces are used for binding to ICL-like single/double-strand junction-containing DNA substrates. A structural model for FAAP24 bound to dsDNA has been made based on homology with the translesion polymerase iota. Site-directed mutagenesis, sequence conservation and charge distribution support the dsDNA-binding model. Analogous to other HhH domain-containing proteins, we suggest that multiple FAAP24 regions together contribute to binding to single/double-strand junction, which could contribute to specificity in ICL DNA recognition.
Asunto(s)
Proteínas de Unión al ADN/química , ADN/metabolismo , Secuencia de Aminoácidos , ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
In non-specific lac headpiece-DNA complexes selective NMR line broadening is observed that strongly depends on length and composition of the DNA fragments. This broadening involves amide protons found in the non-specific lac-DNA structure to be interacting with the DNA phosphate backbone, and can be ascribed to DNA sliding of the protein along the DNA. This NMR exchange broadening has been used to estimate the 1D diffusion constant for sliding along non-specific DNA. The observed 1D diffusion constant of 4×10(-12) cm(2)/s is two orders of magnitude smaller than derived from previous kinetic experiments, but falls in the range of values determined more recently using single molecule methods. This strongly supports the notion that sliding could play at most a minor role in the association kinetics of binding of lac repressor to lac operator and that other processes such as hopping and intersegment transfer contribute to facilitate the DNA recognition process.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Operón Lac , Represoras Lac , Resonancia Magnética Nuclear Biomolecular , Sitios de Unión/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Cinética , Operón Lac/genética , Represoras Lac/química , Represoras Lac/metabolismo , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Mapeo de Interacción de Proteínas , Especificidad por SustratoRESUMEN
Herein, we report a novel strategy to engineer an acid-sensitive anticancer theranostic agent using a vector-drug ensemble. The ensemble was synthesized by directly conjugating the linoleic acid (LA)-modified branched polyethyleneimine with a chemotherapeutic drug trifluorothymidine. Linoleic acid residues were grafted onto 25 kDa polyethyleneimine (PEI) by treating PEI with linoleic acid chloroanhydride. 5-Trifluoromethyl-2'-deoxyuridine (trifluorothymidine, TFT) was introduced into LA-PEI conjugate by phosphorylating the conjugate with amidophosphate of trifluorothymidine 5'-monophosphate (pTFT), which had been activated by its conversion into the N,N-dimethylaminopyridine derivative. The extent of mononucleotide analog incorporation in the polymer was regulated by the ratio of pTFT to the polymer during the synthesis. Samples containing 20-70 TFT residues per PEI molecule were obtained. The cytotoxicity of PEI-LA-pTFT conjugates decreased with increasing nucleotide content, as examined using the MTT method. Due to the presence of fluorine atoms, TFT-based conjugates could be detected directly in the animals by (19)F magnetic resonance imaging. In addition, the presence of the amidophosphate group in PEI-LA-pTFT conjugates allowed their detection by in vivo(31)P NMR spectroscopy. Indeed, the (31)P NMR signal of a phosphoramide (δ ~ 12 ppm) was observed in the mouse muscle tissue treated with PEI-LA-pTFT conjugate along with the signals from endogenous phosphorus-containing compounds. At the same time, the use of PEI-LA-pTFT conjugate for chemotherapeutic drug delivery is limited due to the low release of pTFT from the carrier. To enhance the release of the drug from the conjugate in the endosomes, PEI-LA polymer was coupled with urocanic acid (UA), which bears imidazole ring and thus can form an acid-labile P-N bond with pTFT. The PEI-LA-UA-pTFT conjugate containing 30 residues of UA and 40 residues of pTFT was tested against the murine Krebs-II ascites carcinoma, grown as an ascetic tumor. The intraperitoneal injection of the conjugates resulted in prolongation of the animals' life and to the complete disappearance of the tumor after three injections.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/uso terapéutico , Ácido Linoleico/química , Polietileneimina/análogos & derivados , Trifluridina/química , Trifluridina/uso terapéutico , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Carcinoma Krebs 2/tratamiento farmacológico , Línea Celular Tumoral , Portadores de Fármacos/química , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Trifluridina/administración & dosificación , Trifluridina/farmacocinéticaRESUMEN
NMR micro-imaging technique has been used for the measurement of the water content distribution in lenses of senescence-accelerated OXYS rats and age-matched Wistar rats, as well as for the study of water and phosphate transport in rat lenses. The water content in the lens cortex is significantly higher than in the nucleus; the spatial gradient of the water content becomes steeper with age. No difference in the water content distribution has been found between Wistar and OXYS rat lenses of matching ages, although cataract onset in the OXYS rat lens occurs much earlier due to the enhanced generation of reactive oxygen species associated with oxidative stress. This finding implies that cataract development does not lead to significant changes in water content distribution inside the lens. The water transport in rat lenses slows down with age, and in OXYS lenses it is somewhat faster than in lenses of Wistar rats, probably due to the compensatory response to oxidative stress. The application of (31)P MRI for the monitoring of phosphate penetration into a lens has been performed for the first time. It is found that phosphate transport in a lens is significantly slower than that of water.
Asunto(s)
Agua Corporal/metabolismo , Cristalino/metabolismo , Imagen por Resonancia Magnética , Envejecimiento Prematuro/metabolismo , Animales , Transporte Biológico/fisiología , Catarata/metabolismo , Masculino , Estrés Oxidativo , Fosfatos/metabolismo , Ratas , Ratas WistarRESUMEN
Various hyperpolarization methods are able to enhance the sensitivity of nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) by several orders of magnitude. Among these methods are para-hydrogen-induced polarization (PHIP) and signal amplification by reversible exchange (SABRE), which exploit the strong nuclear alignment of para-hydrogen. Several SABRE experiments have been reported but, so far, it has not been possible to account for the experimentally observed sign and magnetic-field dependence of substrate polarization. Herein, we present an analysis based on level anti-crossings (LACs), which provides a complete understanding of the SABRE effect. The field-dependence of both net and anti-phase polarization is measured for several ligands, which can be reproduced by the theory. The similar SABRE field-dependence for different ligands is also explained. In general, the LAC concept allows complex spin dynamics to be unraveled, and is crucial for optimizing the performance of novel hyperpolarization methods in NMR and MRI techniques.
RESUMEN
The photophysics and photochemistry of kynurenic acid (KNA) and kynurenine yellow (KNY) in neutral aqueous solutions were investigated using time-resolved optical spectroscopy. Both molecules have similar quinoline-like structures, the only difference being the absence of conjugation in the nitrogen containing cycle in KNY. The main channel of S(1) excited state decay in the case of partially-unconjugated KNY is the solvent assisted S(1) â S(0) radiationless transition via intermolecular hydrogen bonds (Φ(IC) = 0.96), whereas, in the case of fully-conjugated KNA, it is intersystem crossing to the triplet state (Φ(T) = 0.82). The major intermediate products of the singlet excited KNY deactivation are the triplet state (Φ(T) = 0.022) and, most probably, the enol form (Φ(enol) = 0.012), which decay with the formation of 2,3-dihydro-4-hydroxyquinoline and 4-hydroxyquinoline, respectively. The results obtained show that KNA and KNY, which are products of the decomposition of the UV filter kynurenine, are significantly more photoactive and less photostable than the parent molecule.
Asunto(s)
Ácido Quinurénico/química , Fotólisis , Quinolonas/química , Agua/química , Oxidación-Reducción , Soluciones , Espectrometría de FluorescenciaRESUMEN
Spin hyperpolarization can be coherently transferred to other nuclei in field-cycling NMR experiments. At low magnetic fields spin polarization is redistributed in a strongly coupled network of spins. Polarization transfer is most efficient at fields where level anti-crossings (LACs) occur for the nuclear spin-states. A further condition is that field switching to the LAC positions is non-adiabatic in order to convert the starting population differences into spin coherences that cause time-dependent mixing of states. The power of this method has been demonstrated by studying transfer of photo-Chemically Induced Dynamic Nuclear Polarization (photo-CIDNP) in N-acetyl-tryptophan. We have investigated the magnetic field dependence and time dependence of coherent CIDNP transfer and directly assessed nuclear spin LACs by studying polarization transfer at specific field positions. The proposed approach based on LACs is not limited to CIDNP but is advantageous for enhancing NMR signals by spin order transfer from any type of hyper-polarized nuclei.
RESUMEN
We propose pulse sequences for Reaction Yield Detected Magnetic Resonance (RYDMR), which are based on refocusing the zero-quantum coherences in radical pairs by non-selective microwave pulses and using the population of a radical pair singlet spin state as an observable. The new experiments are analogues of existing EPR experiments such as the primary echo, Carr-Purcell, ESEEM, stimulated echo and Mims ENDOR. All pulse sequences are supported by analytical results and numerical calculations. The pulse sequences can be used for more efficient and highly detailed characterization of intermediates of chemical reactions and charge carriers in organic semiconductors.
RESUMEN
Effects of spin-spin interactions on the nuclear magnetic relaxation dispersion (NMRD) of protons were studied in a situation where spin ½ hetero-nuclei are present in the molecule. As in earlier works [K. L. Ivanov, A. V. Yurkovskaya, and H.-M. Vieth, J. Chem. Phys. 129, 234513 (2008); S. E. Korchak, K. L. Ivanov, A. V. Yurkovskaya, and H.-M. Vieth, ibid. 133, 194502 (2010)], spin-spin interactions have a pronounced effect on the relaxivity tending to equalize the longitudinal relaxation times once the spins become strongly coupled at a sufficiently low magnetic field. In addition, we have found influence of (19)F nuclei on the proton NMRD, although in the whole field range, studied protons and fluorine spins were only weakly coupled. In particular, pronounced features in the proton NMRD were found; but each feature was predominantly observed only for particular spin states of the hetero-nuclei. The features are explained theoretically; it is shown that hetero-nuclei can affect the proton NMRD even in the limit of weak coupling when (i) protons are coupled strongly and (ii) have spin-spin interactions of different strengths with the hetero-nuclei. We also show that by choosing the proper magnetic field strength, one can selectively transfer proton spin magnetization between spectral components of choice.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Protones , Compuestos de Anilina/químicaRESUMEN
Binding of the nuclear factor-Y complex (NF-Y) to the inverted CCAAT-box interferes with transcription activation through nucleosome reorganization. The three homologous proteins forming the zinc-fingers and homeoboxes (ZHX) family interact with the activation domain of NF-Ya to repress transcription. Each ZHX-protein contains two generic C2H2 zinc-fingers (ZNF1 and ZNF2) followed by five homeodomains. Although the proteins have been related to the occurrence of certain cancers, the function and structure of the individual ZHX domains are still unknown. Here, we determined the structure of the tandem zinc-finger region of human ZHX1. Folding and secondary structure predictions combined with expression screening revealed that the C-terminal extension (E) to ZNF2 could form a single domain with the two hZHX1 zinc-fingers. We therefore decided to determine the solution structure of the zinc-fingers followed by this extension. We show that both zinc-fingers adopt canonical betabetaalpha-folds in which a zinc ion is coordinated by two cysteine and two histidine residues. The C-terminal extension to ZNF2 forms two beta-strands to make a beta-sheet with the beta-strands of this zinc-finger. The ZNF1 and ZNF2-E domains do not show evident contacts and their mutual orientation seems variable. The high degree of sequence conservation among ZHX family members permitted us to prepare homology models for ZHX2 and ZHX3, revealing distinct surface characteristics for each family member. Implications of these structural features for ZHX-functioning in transcription regulation are discussed.
Asunto(s)
Proteínas de Homeodominio/química , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Dedos de Zinc , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Proteínas de Homeodominio/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Factores de Transcripción/genéticaRESUMEN
NMR studies of structural aspects of allosteric regulation by the Lac repressor requires overexpression and isotope labeling of the protein. The size of the repressor makes it a challenging target, putting constraints on both expression conditions and sample preparation methods to overcome problems associated with studies of larger proteins by NMR. We optimized protocols for the production of deuterated functionally active thermostable dimeric Lac repressor and its core domain mutants. The Lac repressor core domain has never been obtained as a recombinant protein, possibly due to the observed toxicity to the host cells. We overcame the core domain induced toxicity by co-expression of this domain with the full length Lac repressor, combined with a stringent control of culture conditions. Significant overexpression was only obtained if during all stages of pre-culturing the bacteria were kept in their exponential growth phase at low density. The sensitivity of NMR measurements is dramatically affected by buffer conditions; we therefore used a thermofluor buffer optimization screen to determine the optimal buffer conditions. The combined thermofluor and NMR screening method yielded thermostable fully functional Lac repressor domain samples suitable for high-resolution NMR studies. The optimized procedures to adapt Escherichia coli to growth in D2O, to overcome toxicity, and to optimize protein sample conditions provides a broad range of universally applicable techniques for production of larger proteins for NMR spectroscopy.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Resonancia Magnética Nuclear Biomolecular/métodos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Represoras/biosíntesis , Amidas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Técnicas de Cultivo de Célula/métodos , Cromatografía de Afinidad , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Concentración de Iones de Hidrógeno , Isopropil Tiogalactósido/química , Isopropil Tiogalactósido/metabolismo , Marcaje Isotópico , Represoras Lac , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Represoras/aislamiento & purificación , Cloruro de Sodio/química , TemperaturaRESUMEN
The emerging antibiotics-resistance problem has underlined the urgent need for novel antimicrobial agents. Lantibiotics (lanthionine-containing antibiotics) are promising candidates to alleviate this problem. Nisin, a member of this family, has a unique pore-forming activity against bacteria. It binds to lipid II, the essential precursor of cell wall synthesis. As a result, the membrane permeabilization activity of nisin is increased by three orders of magnitude. Here we report the solution structure of the complex of nisin and lipid II. The structure shows a novel lipid II-binding motif in which the pyrophosphate moiety of lipid II is primarily coordinated by the N-terminal backbone amides of nisin via intermolecular hydrogen bonds. This cage structure provides a rationale for the conservation of the lanthionine rings among several lipid II-binding lantibiotics. The structure of the pyrophosphate cage offers a template for structure-based design of novel antibiotics.
Asunto(s)
Antibacterianos/metabolismo , Difosfatos/metabolismo , Metabolismo de los Lípidos , Nisina/metabolismoRESUMEN
Human ERCC1/XPF is a structure-specific endonuclease involved in multiple DNA repair pathways. We present the solution structure of the non-catalytic ERCC1 central domain. Although this domain shows structural homology with the catalytically active XPF nuclease domain, functional investigation reveals a completely distinct function for the ERCC1 central domain by performing interactions with both XPA and single-stranded DNA. These interactions are non-competitive and can occur simultaneously through distinct interaction surfaces. Interestingly, the XPA binding by ERCC1 and the catalytic function of XPF are dependent on a structurally homologous region of the two proteins. Although these regions are strictly conserved in each protein family, amino acid composition and surface characteristics are distinct. We discuss the possibility that after XPF gene duplication, the redundant ERCC1 central domain acquired novel functions, thereby increasing the fidelity of eukaryotic DNA repair.
Asunto(s)
ADN de Cadena Simple/química , Proteínas de Unión al ADN/química , Endonucleasas/química , Proteína de la Xerodermia Pigmentosa del Grupo A/química , Secuencia de Aminoácidos , Sitios de Unión , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Dimerización , Endonucleasas/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Terciaria de Proteína , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismoRESUMEN
The human XPF-ERCC1 protein complex plays an essential role in nucleotide excision repair by catalysing positioned nicking of a DNA strand at the 5' side of the damage. We have recently solved the structure of the heterodimeric complex of the C-terminal domains of XPF and ERCC1 (Tripsianes et al., Structure 2005;13:1849-1858). We found that this complex comprises a pseudo twofold symmetry axis and that the helix-hairpin-helix motif of ERCC1 is required for DNA binding, whereas the corresponding domain of XPF is functioning as a scaffold for complex formation with ERCC1. Despite the functional importance of heterodimerization, the C-terminal domain of XPF can also form homodimers in vitro. We here compare the stabilities of homodimeric and heterodimeric complexes of the C-terminal domains of XPF and ERCC1. The higher stability of the XPF HhH complexes under various experimental conditions, determined using CD and NMR spectroscopy and mass spectrometry, is well explained by the structural differences that exist between the HhH domains of the two complexes. The XPF HhH homodimer has a larger interaction interface, aromatic stacking interactions, and additional hydrogen bond contacts as compared to the XPF/ERCC1 HhH complex, which accounts for its higher stability.
Asunto(s)
Proteínas de Unión al ADN/química , Endonucleasas/química , Aminoácidos Aromáticos , Reparación del ADN , Dimerización , Humanos , Enlace de Hidrógeno , Complejos Multiproteicos/química , Unión Proteica , Estructura Terciaria de ProteínaAsunto(s)
Resonancia Magnética Nuclear Biomolecular , Ácidos Nucleicos/metabolismo , Proteínas/metabolismo , Sitios de Unión , ADN/química , ADN/metabolismo , Humanos , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Ácidos Nucleicos/química , Unión Proteica/fisiología , Proteínas/química , ARN/química , ARN/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Proteínas Represoras/fisiología , Activación Transcripcional/fisiologíaRESUMEN
The human ERCC1/XPF complex is a structure-specific endonuclease with defined polarity that participates in multiple DNA repair pathways. We report the heterodimeric structure of the C-terminal domains of both proteins responsible for ERCC1/XPF complex formation. Both domains exhibit the double helix-hairpin-helix motif (HhH)2, and they are related by a pseudo-2-fold symmetry axis. In the XPF domain, the hairpin of the second motif is replaced by a short turn. The ERCC1 domain folds properly only in the presence of the XPF domain, which implies a role for XPF as a scaffold for the folding of ERCC1. The intersubunit interactions are largely hydrophobic in nature. NMR titration data show that only the ERCC1 domain of the ERCC1/XPF complex is involved in DNA binding. On the basis of these findings, we propose a model for the targeting of XPF nuclease via ERCC1-mediated interactions in the context of nucleotide excision repair.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/química , Endonucleasas/química , Secuencias Hélice-Asa-Hélice , Secuencia de Aminoácidos , ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Dimerización , Endonucleasas/fisiología , Humanos , Datos de Secuencia Molecular , Mapeo de Interacción de Proteínas , Estructura Terciaria de ProteínaRESUMEN
The N-terminally truncated variant of photoactive yellow protein (Delta25-PYP) undergoes a very similar photocycle as the corresponding wild-type protein (WT-PYP), although the lifetime of its light-illuminated (pB) state is much longer. This has allowed determination of the structure of both its dark- (pG) as well as its pB-state in solution by nuclear magnetic resonance (NMR) spectroscopy. The pG structure shows a well-defined fold, similar to WT-PYP and the X-ray structure of the pG state of Delta25-PYP. In the long-lived photocycle intermediate pB, the central beta sheet is still intact, as well as a small part of one alpha helix. The remainder of pB is unfolded and highly flexible, as evidenced by results from proton-deuterium exchange and NMR relaxation studies. Thus, the partially unfolded nature of the presumed signaling state of PYP in solution, as suggested previously, has now been structurally demonstrated.