RESUMEN
Histamine receptors are a group of G protein-coupled receptors (GPCRs) that play important roles in various physiological and pathophysiological conditions. Antihistamines that target the histamine H1 receptor (H1R) have been widely used to relieve the symptoms of allergy and inflammation. Here, to uncover the details of the regulation of H1R by the known second-generation antihistamines, thereby providing clues for the rational design of newer antihistamines, we determine the cryo-EM structure of H1R in the apo form and bound to different antihistamines. In addition to the deep hydrophobic cavity, we identify a secondary ligand-binding site in H1R, which potentially may support the introduction of new derivative groups to generate newer antihistamines. Furthermore, these structures show that antihistamines exert inverse regulation by utilizing a shared phenyl group that inserts into the deep cavity and block the movement of the toggle switch residue W4286.48. Together, these results enrich our understanding of GPCR modulation and facilitate the structure-based design of novel antihistamines.
Asunto(s)
Antagonistas de los Receptores Histamínicos H1 , Histamina , Antagonistas de los Receptores Histamínicos H1/farmacología , Antagonistas de los Receptores Histamínicos H1/química , Antagonistas de los Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H1/genética , Receptores Histamínicos H1/metabolismo , Antagonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos/química , Antagonistas de los Receptores Histamínicos/metabolismo , Receptores HistamínicosRESUMEN
The Wnt pathway is an evolutionarily conserved pathway involved in stem cell homeostasis and tissue regeneration. Aberrant signaling in the Wnt pathway is highly associated with cancer. Developing antibodies to block overactivation of Frizzled receptors (FZDs), the main receptors in the Wnt pathway, is one of the viable options for treating cancer. However, obtaining isoform-specific antibodies is often challenging due to the high degree of homology among the ten FZDs. In this study, by using a synthetic library, we identified an antibody named pF8_AC3 that preferentially binds to FZD8. Guided by the structure of the complex of pF8_AC3 and FZD8, a second-generation targeted library was further constructed, and finally, the FZD8-specific antibody sF8_AG6 was obtained. Cell-based assays showed that these antibodies could selectively block FZD8-mediated signaling activation. Taken together, these antibodies have the potential to be developed into therapeutic drugs in the future.
Asunto(s)
Neoplasias , Receptores de Superficie Celular , Humanos , Receptores de Superficie Celular/metabolismo , Vía de Señalización Wnt , Biblioteca de GenesRESUMEN
G-protein-coupled receptors (GPCRs) are a class of integral membrane proteins that detect environmental cues and trigger cellular responses. Deciphering the functional states of GPCRs induced by various ligands has been one of the primary goals in the field. Here we developed an effective universal method for GPCR cryo-electron microscopy structure determination without the need to prepare GPCR-signaling protein complexes. Using this method, we successfully solved the structures of the ß2-adrenergic receptor (ß2AR) bound to antagonistic and agonistic ligands and the adhesion GPCR ADGRL3 in the apo state. For ß2AR, an intermediate state stabilized by the partial agonist was captured. For ADGRL3, the structure revealed that inactive ADGRL3 adopts a compact fold and that large unusual conformational changes on both the extracellular and intracellular sides are required for activation of adhesion GPCRs. We anticipate that this method will open a new avenue for understanding GPCR structureâfunction relationships and drug development.
Asunto(s)
Receptores Adrenérgicos beta 2 , Receptores Acoplados a Proteínas G , Modelos Moleculares , Microscopía por Crioelectrón , Receptores Acoplados a Proteínas G/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , LigandosRESUMEN
Implementation of designer receptors in engineered cells confers them to sense a particular physiological or disease state and respond with user-defined programs. To expand the therapeutic application scope of engineered cells, synthetic receptors realized through different strategies are in great demand. Here, we develop a synthetic receptor system that exerts dual control by incorporating two transmembrane helices for the signal chain. Together with a sensor-actuator device with minimal background signals and a positive loop circuit, this receptor system can sensitively respond to extracellular protein signals. We demonstrate that this synthetic receptor system can be readily adapted to respond to various inputs, such as interleukin-1 (IL-1), programmed death ligand 1 (PD-L1), and HER2, and release customized outputs, including fluorescence signals and the therapeutic molecule IL-2. The robust signaling ability and generality of this receptor system promise it to be a useful tool in the field of cell engineering for fundamental research and translational applications.
Asunto(s)
Receptores Artificiales , Transducción de Señal , Procesamiento Proteico-Postraduccional , Biología SintéticaRESUMEN
The M2 muscarinic receptor (M2R) is a prototypical G-protein-coupled receptor (GPCR) that serves as a model system for understanding GPCR regulation by both orthosteric and allosteric ligands. Here, we investigate the mechanisms governing M2R signaling versatility using cryo-electron microscopy (cryo-EM) and NMR spectroscopy, focusing on the physiological agonist acetylcholine and a supra-physiological agonist iperoxo, as well as a positive allosteric modulator LY2119620. These studies reveal that acetylcholine stabilizes a more heterogeneous M2R-G-protein complex than iperoxo, where two conformers with distinctive G-protein orientations were determined. We find that LY2119620 increases the affinity for both agonists, but differentially modulates agonists efficacy in G-protein and ß-arrestin pathways. Structural and spectroscopic analysis suggest that LY211620 stabilizes distinct intracellular conformational ensembles from agonist-bound M2R, which may enhance ß-arrestin recruitment while impairing G-protein activation. These results highlight the role of conformational dynamics in the complex signaling behavior of GPCRs, and could facilitate design of better drugs.
Asunto(s)
Acetilcolina , Receptores Muscarínicos , Microscopía por Crioelectrón , Regulación Alostérica/fisiología , Receptores Muscarínicos/metabolismo , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Unión al GTP/metabolismo , Ligandos , beta-Arrestinas/metabolismoRESUMEN
The lack of incorporating epitope information into the selection process makes the conventional antibody screening method less effective in identifying antibodies with desired functions. Here, we developed an epitope-directed antibody selection method by designing a directed library favoring the target epitope and a precise "counter" antigen for clearing irrelevant binders in the library. With this method, we successfully isolated an antibody, pF7_A5, that targets the less conserved region on the FZD2/7 CRD as designed. Guided by the structure of pF7_A5-FZD2CRD, a further round of evolution was conducted together with the "counter" antigen selection strategy, and ultimately, an FZD2-specific antibody and an FZD7-preferred antibody were obtained. Because of targeting the predefined functional site, all these antibodies exhibited the expected modulatory activity on the Wnt pathway. Together, the method developed here will be useful in antibody drug discovery, and the identified FZD antibodies will have clinical potential in FZD-related cancer therapy.
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Anticuerpos Monoclonales , Evolución Molecular Dirigida , Mapeo Epitopo , Epítopos , Receptores Frizzled , Vía de Señalización Wnt , Descubrimiento de Drogas , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Receptores Frizzled/química , Receptores Frizzled/genética , Receptores Frizzled/inmunología , Vía de Señalización Wnt/inmunología , Anticuerpos Monoclonales/inmunología , Mapeo Epitopo/métodos , Humanos , Conformación Proteica , Evolución Molecular Dirigida/métodosRESUMEN
The major challenge to controlling the COVID pandemic is the rapid mutation rate of the SARS-CoV-2 virus, leading to the escape of the protection of vaccines and most of the neutralizing antibodies to date. Thus, it is essential to develop neutralizing antibodies with broad-spectrum activity targeting multiple SARS-CoV-2 variants. Here, we report a synthetic nanobody (named C5G2) obtained by phage display and subsequent antibody engineering. C5G2 has a single-digit nanomolar binding affinity to the RBD domain and inhibits its binding to ACE2 with an IC50 of 3.7 nM. Pseudovirus assays indicated that monovalent C5G2 could protect the cells from infection with SARS-CoV-2 wild-type virus and most of the viruses of concern, i.e., Alpha, Beta, Gamma and Omicron variants. Strikingly, C5G2 has the highest potency against Omicron BA.1 among all the variants, with an IC50 of 4.9 ng/mL. The cryo-EM structure of C5G2 in complex with the spike trimer showed that C5G2 binds to RBD mainly through its CDR3 at a conserved region that does not overlap with the ACE2 binding surface. Additionally, C5G2 binds simultaneously to the neighboring NTD domain of the spike trimer through the same CDR3 loop, which may further increase its potency against viral infection. Third, the steric hindrance caused by FR2 of C5G2 could inhibit the binding of ACE2 to RBD as well. Thus, this triple-function nanobody may serve as an effective drug for prophylaxis and therapy against Omicron as well as future variants.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , SARS-CoV-2 , Anticuerpos de Dominio Único , Enzima Convertidora de Angiotensina 2 , Anticuerpos Neutralizantes/farmacología , Anticuerpos Antivirales/farmacología , COVID-19 , SARS-CoV-2/efectos de los fármacos , Anticuerpos de Dominio Único/farmacología , Glicoproteína de la Espiga del CoronavirusRESUMEN
Melatonin receptors (MT1 and MT2 in humans) are family A G protein-coupled receptors that respond to the neurohormone melatonin to regulate circadian rhythm and sleep. Numerous efforts have been made to develop drugs targeting melatonin receptors for the treatment of insomnia, circadian rhythm disorder, and cancer. However, designing subtype-selective melatonergic drugs remains challenging. Here, we report the cryo-EM structures of the MT1-Gi signaling complex with 2-iodomelatonin and ramelteon and the MT2-Gi signaling complex with ramelteon. These structures, together with the reported functional data, reveal that although MT1 and MT2 possess highly similar orthosteric ligand-binding pockets, they also display distinctive features that could be targeted to design subtype-selective drugs. The unique structural motifs in MT1 and MT2 mediate structural rearrangements with a particularly wide opening on the cytoplasmic side. Gi is engaged in the receptor core shared by MT1 and MT2 and presents a conformation deviating from those in other Gi complexes. Together, our results provide new clues for designing melatonergic drugs and further insights into understanding the G protein coupling mechanism.
Asunto(s)
Receptor de Melatonina MT1/química , Receptor de Melatonina MT2/química , Secuencias de Aminoácidos , Microscopía por Crioelectrón , Humanos , Indenos/química , Indenos/metabolismo , Ligandos , Melatonina/análogos & derivados , Melatonina/química , Melatonina/metabolismo , Unión Proteica , Conformación Proteica , Receptor de Melatonina MT1/genética , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/genética , Receptor de Melatonina MT2/metabolismoRESUMEN
A method for fast and highly sensitive detection of antibodies in serum would greatly facilitate the early diagnosis of disease and infection and dose optimization of therapeutic antibody. Bioluminescence detection with LUMABS (renamed mNeonG-LUMABS, where mNeonG is short for mNeonGreen) sensors based on bioluminescence resonance energy transfer (BRET) between blue-emitting luciferase Nluc and green fluorescent protein (FP) mNeonGreen has been demonstrated to enable fast detection of antibodies directly in serum with reasonable sensitivity. However, some mNeonG-LUMABS sensors exhibit low sensitivity, and thus, sensitivity improvement remains imperative. Here, we report a bright green FP, Clover4, obtained by structure-guided mutagenesis of green FP Clover. Despite similar brightness and fluorescence spectra of Clover and mNeonGreen, Clover4-LUMABS sensors exhibit a largely increased dynamic range (maximum 20-fold) and much lower limit of detection (LOD) (maximum 5.6-fold), most likely because Clover4 is positioned in a more parallel orientation to Nluc in LUMABS. Due to modular design, Clover4-LUMABS offers a general BRET system for fast and highly sensitive antibody detection in serum.
Asunto(s)
Anticuerpos , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Luminiscentes/genética , Transferencia Resonante de Energía de Fluorescencia/métodos , Límite de DetecciónRESUMEN
Two-component systems (TCS), which typically consist of a membrane-embedded histidine kinase and a cytoplasmic response regulator, are the dominant signaling proteins for transduction of environmental stimuli into cellular response pathways in prokaryotic cells. HptRSA is a recently identified TCS consisting of the G6P-associated sensor protein (HptA), transmembrane histidine kinase (HptS), and cytoplasmic effector (HptR). HptRSA mediates glucose-6-phosphate (G6P) uptake to support Staphylococcus aureus growth and multiplication within various host cells. How the mechanism by which HptRSA perceives G6P and triggers a downstream response has remained elusive. Here, we solved the HptA structures in apo and G6P-bound states. G6P binding in the cleft between two HptA domains caused a conformational closing movement. The solved structures of HptA in complex with the periplasmic domain of HptS showed that HptA interacts with HptS through both constitutive and switchable interfaces. The G6P-free form of HptA binds to the membrane-distal side of the HptS periplasmic domain (HptSp), resulting in a parallel conformation of the HptSp protomer pair. However, once HptA associates with G6P, its intramolecular domain closure switches the HptA-HptSp contact region into the membrane-proximal domain, which causes rotation and closure of the C termini of each HptSp protomer. Through biochemical and growth assays of HptA and HptS mutant variants, we proposed a distinct mechanism of interface switch-mediated signaling transduction. Our results provide mechanistic insights into bacterial nutrient sensing and expand our understanding of the activation modes by which TCS communicates external signals.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Histidina Quinasa/química , Histidina Quinasa/metabolismo , Transducción de Señal , Fenómenos Fisiológicos Bacterianos , Ligandos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-ActividadRESUMEN
Secreted Wnt proteins regulate development and adult tissue homeostasis by binding and activating cell-surface Frizzled receptors and co-receptors including LRP5/6. The hydrophobicity of Wnt proteins has complicated their purification and limited their use in basic research and as therapeutics. We describe modular tetravalent antibodies that can recruit Frizzled and LRP5/6 in a manner that phenocopies the activities of Wnts both in vitro and in vivo. The modular nature of these synthetic Frizzled and LRP5/6 Agonists, called FLAgs, enables tailored engineering of specificity for one, two or multiple members of the Frizzled family. We show that FLAgs underlie differentiation of pluripotent stem cells, sustain organoid growth, and activate stem cells in vivo. Activation of Wnt signaling circuits with tailored FLAgs will enable precise delineation of functional outcomes directed by distinct receptor combinations and could provide a new class of therapeutics to unlock the promise of regenerative medicine.
Asunto(s)
Anticuerpos/metabolismo , Receptores Frizzled/agonistas , Vía de Señalización Wnt/efectos de los fármacos , Animales , Línea Celular , Humanos , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad/agonistas , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/agonistas , Ratones , Organoides/efectos de los fármacos , Organoides/crecimiento & desarrollo , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/fisiología , Unión ProteicaRESUMEN
Eukaryotes rely on efficient distribution of energy and carbon skeletons between organs in the form of sugars. Glucose in animals and sucrose in plants serve as the dominant distribution forms. Cellular sugar uptake and release require vesicular and/or plasma membrane transport proteins. Humans and plants use proteins from three superfamilies for sugar translocation: the major facilitator superfamily (MFS), the sodium solute symporter family (SSF; only in the animal kingdom), and SWEETs. SWEETs carry mono- and disaccharides across vacuolar or plasma membranes. Plant SWEETs play key roles in sugar translocation between compartments, cells, and organs, notably in nectar secretion, phloem loading for long distance translocation, pollen nutrition, and seed filling. Plant SWEETs cause pathogen susceptibility possibly by sugar leakage from infected cells. The vacuolar Arabidopsis thaliana AtSWEET2 sequesters sugars in root vacuoles; loss-of-function mutants show increased susceptibility to Pythium infection. Here we show that its orthologue, the vacuolar glucose transporter OsSWEET2b from rice (Oryza sativa), consists of an asymmetrical pair of triple-helix bundles, connected by an inversion linker transmembrane helix (TM4) to create the translocation pathway. Structural and biochemical analyses show OsSWEET2b in an apparent inward (cytosolic) open state forming homomeric trimers. TM4 tightly interacts with the first triple-helix bundle within a protomer and mediates key contacts among protomers. Structure-guided mutagenesis of the close paralogue SWEET1 from Arabidopsis identified key residues in substrate translocation and protomer crosstalk. Insights into the structure-function relationship of SWEETs are valuable for understanding the transport mechanism of eukaryotic SWEETs and may be useful for engineering sugar flux.
Asunto(s)
Proteínas Facilitadoras del Transporte de la Glucosa/química , Oryza/química , Proteínas de Plantas/química , Multimerización de Proteína , Arabidopsis/química , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Cristalografía por Rayos X , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Proteínas de Transporte de Monosacáridos/química , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Oryza/genética , Floema , Proteínas de Plantas/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
SWEETs and their prokaryotic homologues are monosaccharide and disaccharide transporters that are present from Archaea to plants and humans. SWEETs play crucial roles in cellular sugar efflux processes: that is, in phloem loading, pollen nutrition and nectar secretion. Their bacterial homologues, which are called SemiSWEETs, are among the smallest known transporters. Here we show that SemiSWEET molecules, which consist of a triple-helix bundle, form symmetrical, parallel dimers, thereby generating the translocation pathway. Two SemiSWEET isoforms were crystallized, one in an apparently open state and one in an occluded state, indicating that SemiSWEETs and SWEETs are transporters that undergo rocking-type movements during the transport cycle. The topology of the triple-helix bundle is similar yet distinct to that of the basic building block of animal and plant major facilitator superfamily (MFS) transporters (for example, GLUTs and SUTs). This finding indicates two possibilities: that SWEETs and MFS transporters evolved from an ancestral triple-helix bundle or that the triple-helix bundle represents convergent evolution. In SemiSWEETs and SWEETs, two triple-helix bundles are arranged in a parallel configuration to produce the 6- and 6 + 1-transmembrane-helix pores, respectively. In the 12-transmembrane-helix MFS transporters, four triple-helix bundles are arranged into an alternating antiparallel configuration, resulting in a much larger 2 × 2 triple-helix bundle forming the pore. Given the similarity of SemiSWEETs and SWEETs to PQ-loop amino acid transporters and to mitochondrial pyruvate carriers (MPCs), the structures characterized here may also be relevant to other transporters in the MtN3 clan. The insight gained from the structures of these transporters and from the analysis of mutations of conserved residues will improve the understanding of the transport mechanism, as well as allow comparative studies of the different superfamilies involved in sugar transport and the evolution of transporters in general.
Asunto(s)
Proteínas Bacterianas/química , Leptospira/química , Proteínas de Transporte de Monosacáridos/química , Vibrio/química , Arabidopsis/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Evolución Molecular , Glucosa/metabolismo , Leptospira/genética , Modelos Moleculares , Proteínas de Transporte de Monosacáridos/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Movimiento , Conformación Proteica , Multimerización de Proteína , Relación Estructura-ActividadRESUMEN
Human CENP-N and CENP-L have been reported to selectively recognize the CENP-A nucleosome and to contribute to recruiting other constitutive centromere-associated network (CCAN) complexes involved in assembly of the inner kinetochore. As their homologues, Chl4 and Iml3 from budding yeast function in a similar way in de novo assembly of the kinetochore. A lack of biochemical and structural information precludes further understanding of their exact role at the molecular level. Here, the crystal structure of Iml3 is presented and the structure shows that Iml3 adopts an elongated conformation with a series of intramolecular interactions. Pull-down assays revealed that the C-terminal domain of Chl4, which forms a dimer in solution, is responsible for Iml3 binding. Acting as a heterodimer, the Chl4-Iml3 complex exhibits a low-affinity nonspecific DNA-binding activity which may play an important role in the kinetochore-assembly process.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Cristalografía por Rayos X , Proteínas del Citoesqueleto/química , Células HeLa , Humanos , Cinetocoros/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Mapeo de Interacción de Proteínas , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Mannonate dehydratase (ManD; EC4.2.1.8) catalyzes the dehydration of D-mannonate to 2-keto-3-deoxygluconate. It is the third enzyme in the pathway for dissimilation of D-glucuronate to 2-keto-3-deoxygluconate involving in the Entner-Doudoroff pathway in certain bacterial and archaeal species. ManD from Gram negative bacteria has an insert sequence as compared to those from Gram positives revealed by sequence analysis. To evaluate the impact of this insert sequence on the catalytic efficiency, we solved the crystal structures of ManD from Escherichia coli strain K12 and its complex with D-mannonate, which reveal that this insert sequence forms two α helices locating above the active site. The two insert α helices introduce a loop that forms a cap covering the substrate binding pocket, which restricts the tunnels of substrate entering and product releasing from the active site. Site-directed mutations and enzymatic activity assays confirm that the catalytic rate is decreased by this loop. These features are conserved among Gram negative bacteria. Thus, the insert sequence of ManD from Gram negative bacteria acts as a common inducer to decrease the catalytic rate and consequently the glucuronate metabolic rate as compared to those from Gram positives. Moreover, residues essential for substrate to enter the active site were characterized via structural analysis and enzymatic activity assays.
Asunto(s)
Bacterias Gramnegativas/enzimología , Hidroliasas/química , Hidroliasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Escherichia coli/enzimología , Gluconatos/metabolismo , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Azúcares Ácidos/metabolismo , Difracción de Rayos XRESUMEN
The yeast Shu complex, consisting of the proteins Shu1, Shu2, Psy3, and Csm2, maintains genomic stability by coupling post-replication repair to homologous recombination. However, a lack of biochemical and structural information on the Shu proteins precludes revealing their precise roles within the pathway. Here, we report on the 1.9-Å crystal structure of the Psy3-Csm2 complex. The crystal structure shows that Psy3 forms a heterodimer with Csm2 mainly through a hydrophobic core. Unexpectedly, Psy3 and Csm2 share a similar architecture that closely resembles the ATPase core domain of Rad51. The L2 loop present in Psy3 and Csm2 is similar to that of Rad51 and confers the DNA binding activity of the Shu complex. As with Rad51, the Shu complex appears to form a nucleoprotein filament by binding nonspecifically to DNA. Structure-based mutagenesis studies have demonstrated that the DNA binding activity of the Shu complex is essential for repair of the methyl methanesulfonate-induced DNA damage. Our findings provide good foundations for the understanding of the Srs2 regulation by the Shu complex.
Asunto(s)
ADN de Hongos/química , Proteínas de Unión al ADN/química , Complejos Multiproteicos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Cristalografía por Rayos X , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-ActividadRESUMEN
Fanconi anaemia is a rare genetic disease characterized by chromosomal instability and cancer susceptibility. The Fanconi anaemia complementation group protein M (FANCM) forms an evolutionarily conserved DNA-processing complex with MHF1/MHF2 (histone-fold-containing proteins), which is essential for DNA repair in response to genotoxic stress. Here we present the crystal structures of the MHF1-MHF2 complex alone and bound to a fragment of FANCM (FANCM(661-800), designated FANCM-F). The structures show that MHF1 and MHF2 form a compact tetramer to which FANCM-F binds through a 'dual-V' shaped structure. FANCM-F and (MHF1-MHF2)(2) cooperate to constitute a new DNA-binding site that is coupled to the canonical L1L2 region. Perturbation of the MHF-FANCM-F structural plasticity changes the localization of FANCM in vivo. The MHF-FANCM interaction and its subcellular localization are altered by a disease-associated mutant of FANCM. These findings reveal the molecular basis of MHF-FANCM recognition and provide mechanistic insights into the pathway leading to Fanconi anaemia.