RESUMEN
Overexpression of human epidermal growth factor receptor 2 (HER2) in breast and gastric cancers is associated with a poor prognosis, making it an important therapeutic target. Here, we establish a novel cancer-specific anti-HER2 antibody, H2Mab-214. H2Mab-214 reacts with HER2 on cancer cells, but unlike the therapeutic antibody trastuzumab, it does not react with HER2 on normal cells in flow cytometry measurements. A crystal structure suggests that H2Mab-214 recognizes a structurally disrupted region in the HER2 domain IV, which normally forms a ß-sheet. We show that this misfolding is inducible by site-directed mutagenesis mimicking the disulfide bond defects that also may occur in cancer cells, indicating that the local misfolding in the Cys-rich domain IV governs the cancer-specificity of H2Mab-214. Furthermore, we show that H2Mab-214 effectively suppresses tumor growth in xenograft mouse models. Our findings offer a potential strategy for developing cancer-specific therapeutic antibodies that target partially misfolded cell surface receptors.
Asunto(s)
Pliegue de Proteína , Receptor ErbB-2 , Ensayos Antitumor por Modelo de Xenoinjerto , Receptor ErbB-2/metabolismo , Receptor ErbB-2/química , Humanos , Animales , Ratones , Línea Celular Tumoral , Modelos Moleculares , Femenino , Cristalografía por Rayos X , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/química , Trastuzumab/farmacología , Trastuzumab/química , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/tratamiento farmacológicoRESUMEN
Celastrol, a bioactive molecule extracted from the Tripterygium wilfordii plant, has been shown to exhibit anti-inflammatory properties. However, its mechanism of action has not been fully elucidated. Here, we show that celastrol suppresses humoral immune responses and autoimmunity by disabling a protein complex consisting of copper metabolism MURR1 domain-containing (COMMD) 3 and COMMD8 (COMMD3/8 complex), a signaling adaptor for chemoattractant receptors. Having demonstrated the involvement of the COMMD3/8 complex in a mouse model of rheumatoid arthritis, we identified celastrol as a compound that covalently bound to and dissociated the COMMD3/8 complex. Celastrol inhibited B cell migration, reduced antibody responses, and blocked arthritis progression, recapitulating deficiency of the COMMD3/8 complex. These effects of celastrol were abolished in mice expressing a celastrol-resistant mutant of the COMMD3/8 complex. These findings establish that celastrol exerts immunosuppressive activity by targeting the COMMD3/8 complex. Our study suggests that the COMMD3/8 complex is a potentially druggable target in autoimmune diseases and points to celastrol as a lead pharmacologic candidate in this capacity.
Asunto(s)
Enfermedades Autoinmunes , Inmunidad Humoral , Ratones , Animales , Autoinmunidad , Triterpenos PentacíclicosRESUMEN
Image reconstruction by integrating exchangeable single-molecule localization (IRIS) achieves multiplexed super-resolution imaging by high-density labeling with fast exchangeable fluorescent probes. However, previous methods to develop probes for individual targets required a great amount of time and effort. Here, we introduce a method for generating recombinant IRIS probes with a new mutagenesis strategy that can be widely applied to existing antibody sequences. Several conserved tyrosine residues at the base of complementarity-determining regions were identified as candidate sites for site-directed mutagenesis. With a high probability, mutations at candidate sites accelerated the off rate of recombinant antibody-based probes without compromising specific binding. We were able to develop IRIS probes from five monoclonal antibodies and three single-domain antibodies. We demonstrate multiplexed localization of endogenous proteins in primary neurons that visualizes small synaptic connections with high binding density. It is now practically feasible to generate fast-dissociating fluorescent probes for multitarget super-resolution imaging.
Asunto(s)
Colorantes Fluorescentes , Proteínas , Microscopía Fluorescente/métodos , Colorantes Fluorescentes/química , Anticuerpos , Fragmentos de InmunoglobulinasRESUMEN
Signaling by single-pass transmembrane receptors often involves a formation of ligand-induced receptor dimers with particular conformation, and bivalent receptor binders can modulate receptor functions by inducing different receptor dimer conformations, although such agents are difficult to design. Here, we describe the generation of both antagonistic and agonistic receptor dimerizers toward PlexinB1 (PlxnB1), a receptor for semaphorin 4D (Sema4D), by grafting two different PlxnB1-binding peptides onto the human immunoglobulin G1 (IgG1) Fc protein. The function-modulating activity of a peptide Fc was strongly dependent on the type of the peptide as well as the grafting site, with the best variants showing activity at an nM concentration range. Structural analysis of each peptide-PlxnB1 complex revealed that the agonistic Fc dimerizes PlxnB1 in a face-to-face fashion similar to that induced by Sema4D, whereas antagonistic Fc would induce signaling-incompetent PlxnB1 dimer conformation, enforcing the idea that plexin activation is primarily controlled by the receptor orientation within the dimer.
Asunto(s)
Receptores de Superficie Celular , Semaforinas , Proteínas Activadoras de GTPasa , Humanos , Inmunoglobulina G , Ligandos , Péptidos , Receptores de Superficie Celular/metabolismo , Receptores Fc , Semaforinas/genética , Semaforinas/metabolismoRESUMEN
Decoy receptor proteins that trick viruses to bind to them should be resistant to viral escape because viruses that require entry receptors cannot help but bind decoy receptors. Angiotensin-converting enzyme 2 (ACE2) is the major receptor for coronavirus cell entry. Recombinant soluble ACE2 was previously developed as a biologic against acute respiratory distress syndrome (ARDS) and verified to be safe in clinical studies. The emergence of COVID-19 reignited interest in soluble ACE2 as a potential broad-spectrum decoy receptor against coronaviruses. In this review, we summarize recent developments in preclinical studies using various high-affinity mutagenesis and Fc fusion approaches to achieve therapeutic efficacy of recombinant ACE2 decoy receptor against coronaviruses. We also highlight the relevance of stimulating effector immune cells through Fc-receptor engagement and the potential of using liquid aerosol delivery of ACE2 decoy receptors for defense against ACE2-utilizing coronaviruses.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , Tratamiento Farmacológico de COVID-19 , Receptores Virales , Humanos , Unión Proteica , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
The Omicron (B.1.1.529) SARS-CoV-2 variant contains an unusually high number of mutations in the spike protein, raising concerns of escape from vaccines, convalescent serum, and therapeutic drugs. Here, we analyzed the degree to which Omicron pseudo-virus evades neutralization by serum or therapeutic antibodies. Serum samples obtained 3 months after two doses of BNT162b2 vaccination exhibited 18-fold lower neutralization titers against Omicron than parental virus. Convalescent serum samples from individuals infected with the Alpha and Delta variants allowed similar frequencies of Omicron breakthrough infections. Domain-wise analysis using chimeric spike proteins revealed that this efficient evasion was primarily achieved by mutations clustered in the receptor binding domain but that multiple mutations in the N-terminal domain contributed as well. Omicron escaped a therapeutic cocktail of imdevimab and casirivimab, whereas sotrovimab, which targets a conserved region to avoid viral mutation, remains effective. Angiotensin-converting enzyme 2 (ACE2) decoys are another virus-neutralizing drug modality that are free, at least in theory, from complete escape. Deep mutational analysis demonstrated that an engineered ACE2 molecule prevented escape for each single-residue mutation in the receptor binding domain, similar to immunized serum. Engineered ACE2 neutralized Omicron comparably to the Wuhan strain and also showed a therapeutic effect against Omicron infection in hamsters and human ACE2 transgenic mice. Similar to previous SARS-CoV-2 variants, some sarbecoviruses showed high sensitivity against engineered ACE2, confirming the therapeutic value against diverse variants, including those that are yet to emerge.
Asunto(s)
Enzima Convertidora de Angiotensina 2 , COVID-19 , Animales , Anticuerpos Monoclonales Humanizados , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Vacuna BNT162 , COVID-19/terapia , Humanos , Inmunización Pasiva , Ratones , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2 , Sueroterapia para COVID-19RESUMEN
SARS-CoV-2 has mutated during the global pandemic leading to viral adaptation to medications and vaccinations. Here we describe an engineered human virus receptor, ACE2, by mutagenesis and screening for binding to the receptor binding domain (RBD). Three cycles of random mutagenesis and cell sorting achieved sub-nanomolar affinity to RBD. Our structural data show that the enhanced affinity comes from better hydrophobic packing and hydrogen-bonding geometry at the interface. Additional disulfide mutations caused the fixing of a closed ACE2 conformation to avoid off-target effects of protease activity, and also improved structural stability. Our engineered ACE2 neutralized SARS-CoV-2 at a 100-fold lower concentration than wild type; we also report that no escape mutants emerged in the co-incubation after 15 passages. Therapeutic administration of engineered ACE2 protected hamsters from SARS-CoV-2 infection, decreased lung virus titers and pathology. Our results provide evidence of a therapeutic potential of engineered ACE2.
Asunto(s)
Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/farmacología , Tratamiento Farmacológico de COVID-19 , Mutación , SARS-CoV-2/efectos de los fármacos , Glicoproteína de la Espiga del Coronavirus/metabolismo , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/metabolismo , COVID-19/virología , Células Cultivadas , Cricetinae , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Humanos , Masculino , Simulación de Dinámica Molecular , Unión Proteica , Ingeniería de Proteínas/métodos , SARS-CoV-2/aislamiento & purificación , SARS-CoV-2/metabolismoRESUMEN
Advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed by non-enzymatic reaction between reducing-sugar and Arg/Lys in proteins and are involved in various diabetic complications. GA-pyridine is derived from glycolaldehyde and is one of the most cytotoxic AGEs. Here, we established a single-chain Fv (scFv) antibody against GA-pyridine, 73MuL9-scFv, and examined the details of its specificity and antigen recognition by using various techniques involving biophysics, chemical biology and structural biology. We also synthesized several compounds that differ slightly in regard to the position and number of GA-pyridine substituent groups, and revealed that GA-pyridine was specifically bound to 73MuL9-scFv. Thermodynamic analysis revealed that the association of GA-pyridine to 73MuL9-scFv was an exothermic and enthalpy driven reaction, and thus that the antigen recognition involved multiple specific interactions. Crystallographic analysis of the Fv fragment of 73MuL9-scFv revealed that several CH-π and hydrogen bond interactions took place between the Fv-fragment and GA-pyridine, which was consistent with the results of thermodynamic analysis. Further studies using 73MuL9-scFv as a tool to clarify the relevance of GA-pyridine to diabetic complications are warranted.
Asunto(s)
Productos Finales de Glicación Avanzada/inmunología , Piridinas/inmunología , Anticuerpos de Cadena Única/metabolismo , Acetaldehído/análogos & derivados , Acetaldehído/química , Acetaldehído/inmunología , Secuencia de Aminoácidos , Antígenos/química , Antígenos/metabolismo , Biofisica , Cristalografía/métodos , Productos Finales de Glicación Avanzada/química , Humanos , Enlace de Hidrógeno , Piridinas/química , Anticuerpos de Cadena Única/química , TermodinámicaRESUMEN
Recognition of laminin by integrin receptors is central to the epithelial cell adhesion to basement membrane, but the structural background of this molecular interaction remained elusive. Here, we report the structures of the prototypic laminin receptor α6ß1 integrin alone and in complex with three-chain laminin-511 fragment determined via crystallography and cryo-electron microscopy, respectively. The laminin-integrin interface is made up of several binding sites located on all five subunits, with the laminin γ1 chain C-terminal portion providing focal interaction using two carboxylate anchor points to bridge metal-ion dependent adhesion site of integrin ß1 subunit and Asn189 of integrin α6 subunit. Laminin α5 chain also contributes to the affinity and specificity by making electrostatic interactions with large surface on the ß-propeller domain of α6, part of which comprises an alternatively spliced X1 region. The propeller sheet corresponding to this region shows unusually high mobility, suggesting its unique role in ligand capture.
Asunto(s)
Células Epiteliales/metabolismo , Integrina alfa6/metabolismo , Integrina alfa6beta1/metabolismo , Integrina beta1/metabolismo , Laminina/metabolismo , Secuencia de Aminoácidos , Membrana Basal/metabolismo , Sitios de Unión/fisiología , Adhesión Celular/fisiología , Microscopía por Crioelectrón , Cristalografía por Rayos X , Humanos , Conformación Proteica , Dominios Proteicos/fisiología , Electricidad EstáticaRESUMEN
Protein engineering has great potential for devising multifunctional recombinant proteins to serve as next-generation protein therapeutics, but it often requires drastic modifications of the parental protein scaffolds e.g., additional domains at the N/C-terminus or replacement of a domain by another. A discovery platform system, called RaPID (Random non-standard Peptides Integrated Discovery) system, has enabled rapid discovery of small de novo macrocyclic peptides that bind a target protein with high binding specificity and affinity. Capitalizing on the optimized binding properties of the RaPID-derived peptides, here we show that RaPID-derived pharmacophore sequences can be readily implanted into surface-exposed loops on recombinant proteins and maintain both the parental peptide binding function(s) and the host protein function. We refer to this protein engineering method as lasso-grafting and demonstrate that it can endow specific binding capacity toward various receptors into a diverse set of scaffolds that includes IgG, serum albumin, and even capsid proteins of adeno-associated virus, enabling us to rapidly formulate and produce bi-, tri-, and even tetra-specific binder molecules.
Asunto(s)
Péptidos/química , Péptidos/farmacología , Ingeniería de Proteínas/métodos , Proteínas de la Cápside/química , Proteínas Portadoras/química , Línea Celular , Dependovirus , Humanos , Inmunoglobulina G/química , Modelos Moleculares , Albúmina Sérica/química , Bibliotecas de Moléculas PequeñasRESUMEN
Small-angle neutron scattering (SANS) and small- angle X-ray scattering (SAXS) are powerful techniques for the structural characterization of biomolecular complexes. In particular, SANS enables a selective observation of specific components in complexes by selective deuteration with contrast-matching techniques. In most cases, however, biomolecular interaction systems with heterogeneous oligomers often contain unfavorable aggregates and unbound species, hampering data interpretation. To overcome these problems, SAXS has been recently combined with size exclusion chromatography (SEC), which enables the isolation of the target complex in a multi-component system. By contrast, SEC-SANS is only at a preliminary stage. Hence, we herein perform a feasibility study of this method based on our newly developed inverse contrast-matching (iCM) SANS technique using antibody interactions as model systems. Immunoglobulin G (IgG) or its Fc fragment was mixed with 75% deuterated Fc-binding proteins, i.e. a mutated form of IgG-degrading enzyme of Streptococcus pyogenes and a soluble form of Fcγ receptor IIIb, and subjected to SEC-SANS as well as SEC-SAXS as reference. We successfully observe SANS from the non-deuterated IgG or Fc formed in complex with these binding partners, which were unobservable in terms of SANS in D2O, hence demonstrating the potential utility of the SEC-iCM-SANS approach.
Asunto(s)
Cromatografía en Gel/métodos , Fragmentos Fc de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Dispersión del Ángulo Pequeño , Streptococcus pyogenes/metabolismo , Difracción de Rayos X/métodos , Proteínas Portadoras/metabolismo , Estudios de Factibilidad , Modelos Biológicos , Streptococcus pyogenes/inmunologíaRESUMEN
The MAP tag system comprises a 14-residue peptide derived from mouse podoplanin and its high-affinity monoclonal antibody PMab-1. We determined the crystal structure of PMab-1 complexed with the MAP tag peptide and found that the recognition required only the N-terminal 8 residues of MAP tag sequence, enabling the shortening of the tag length without losing the affinity for PMab-1. Furthermore, the structure illustrated that the MAP tag adopts a U-shaped conformation when bound by PMab-1, suggesting that loop-inserted MAP tag would assume conformation compatible with the PMab-1 binding. We inserted the 8-residue MAP tag into multiple loop regions in various proteins including fibronectin type III domain and G-protein-coupled receptors and tested if they maintain PMab-1 reactivity. Despite the conformational restraints forced by the insertion position, all MAP-inserted mutants were expressed well in mammalian cells at levels comparable to the non-tagged proteins. Furthermore, the binding by PMab-1 was fully maintained even for the mutant where MAP tag was inserted at a structurally restricted ß-hairpin, indicating that the MAP tag system has unique feature that allows placement in the middle of protein domain at desired locations. Our results indicate the versatile utility of the MAP tag system in 'site-specific epitope insertion' application.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Mapeo Epitopo/métodos , Péptidos/inmunología , Proteínas Recombinantes/inmunología , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Células Cultivadas , Cristalografía por Rayos X , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Ratones , Péptidos/química , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Wnt signaling plays fundamental roles in organogenesis, tissue regeneration and cancer, but high-resolution structural information of mammalian Wnt proteins is lacking. We solved a 2.8-Å resolution crystal structure of human Wnt3 in complex with mouse Frizzled 8 Cys-rich domain (CRD). Wnt3 grabs the receptor in a manner very similar to that found in Xenopus Wnt8 complexed with the same receptor. Unlike Xenopus Wnt8-bound CRD, however, Wnt3-bound CRD formed a symmetrical dimer in the crystal by exchanging the tip of the unsaturated acyl chain attached to each Wnt3, confirming the ability of Wnt and Frizzled CRD to form a 2:2 complex. The hypervariable 'linker' region of Wnt3 formed a ß-hairpin protrusion opposite from the Frizzled binding interface, consistent with its proposed role in the coreceptor recognition. Direct binding between this segment and the Wnt coreceptor LRP6 was confirmed, enabling us to build a structural model of the Wnt-Frizzled-LRP6 ternary complex.
Asunto(s)
Receptores Acoplados a Proteínas G/química , Proteína Wnt3/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones , Conformación Proteica , Receptores Acoplados a Proteínas G/metabolismo , Proteína Wnt3/metabolismo , XenopusRESUMEN
This corrects the article DOI: 10.1038/nature24994.
RESUMEN
Several gene fusion technologies have been successfully applied to label particular subunits or domains within macromolecular complexes to enable positional mapping of electron microscopy (EM) density maps, but exogenous fusion of a protein domain into the target polypeptide can cause unwanted structural and functional outcomes. Fab fragments from antibodies can be used as labeling reagents during EM visualization without gene manipulation of the target protein, but this method requires a panel of high-affinity antibodies that recognize a wide variety of epitopes. Linear peptide tags and their anti-tag antibodies can be used but they have a limited mapping ability as their placement is usually limited to the terminal regions of a protein. The PA dodecapeptide epitope tag (GVAMPGAEDDVV), forms a tight ß-turn in the antigen binding pocket of its antibody (NZ-1). This capability allows for insertion of the PA tag into various surface-exposed loops within a multi-domain cell adhesion receptor, αIIbß3 integrin. We confirmed that the purified PA-tagged integrin ectodomain fragments can form a stable complex with NZ-1 Fab. Negative stain EM of the various integrin-NZ-1 complexes revealed that a majority of the particles exhibited a clear density corresponding to the NZ-1 Fab; and the positions of the bound Fab were in good agreement with the predicted location of the inserted PA tag. The high-affinity and insertion-compatibility of the PA tag system allowed us to develop a new EM labeling methodology applicable to proteins for which good antibodies are not available.
Asunto(s)
Epítopos/química , Integrinas/química , Microscopía Electrónica/métodos , Proteínas/química , Epítopos/metabolismo , Fragmentos Fab de Inmunoglobulinas/química , Sustancias Macromoleculares/química , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/inmunología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Dominios ProteicosRESUMEN
Malaria is among the most serious infectious diseases affecting humans, accounting for approximately half a million deaths each year. Plasmodium falciparum causes most life-threatening cases of malaria. Acquired immunity to malaria is inefficient, even after repeated exposure to P. falciparum, but the immune regulatory mechanisms used by P. falciparum remain largely unknown. Here we show that P. falciparum uses immune inhibitory receptors to achieve immune evasion. RIFIN proteins are products of a polymorphic multigene family comprising approximately 150-200 genes per parasite genome that are expressed on the surface of infected erythrocytes. We found that a subset of RIFINs binds to either leucocyte immunoglobulin-like receptor B1 (LILRB1) or leucocyte-associated immunoglobulin-like receptor 1 (LAIR1). LILRB1-binding RIFINs inhibit activation of LILRB1-expressing B cells and natural killer (NK) cells. Furthermore, P. falciparum-infected erythrocytes isolated from patients with severe malaria were more likely to interact with LILRB1 than erythrocytes from patients with non-severe malaria, although an extended study with larger sample sizes is required to confirm this finding. Our results suggest that P. falciparum has acquired multiple RIFINs to evade the host immune system by targeting immune inhibitory receptors.
Asunto(s)
Evasión Inmune/inmunología , Receptor Leucocitario Tipo Inmunoglobulina B1/inmunología , Proteínas de la Membrana/inmunología , Plasmodium falciparum/inmunología , Proteínas Protozoarias/inmunología , Receptores Inmunológicos/inmunología , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Células CHO , Cricetulus , Eritrocitos/inmunología , Eritrocitos/parasitología , Células HEK293 , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Receptor Leucocitario Tipo Inmunoglobulina B1/química , Ligandos , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Malaria Falciparum/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Receptores Inmunológicos/química , Tamaño de la MuestraRESUMEN
The evolutional process of disease-associated autoantibodies in systemic lupus erythematosus (SLE) remains to be established. Here we show intraclonal diversification and affinity maturation of anti-nuclear antibody (ANA)-producing B cells in SLE. We identified a panel of monoclonal ANAs recognizing nuclear antigens, such as double-stranded DNA (dsDNA) and ribonucleoproteins (RNPs) from acute SLE subjects. These ANAs had relatively few, but nonetheless critical mutations. High-throughput immunoglobulin sequencing of blood lymphocytes disclosed the existence of sizable ANA lineages shearing critical mutations intraclonally. We further focused on anti-DNA antibodies, which are capable to bind to both single-stranded (ss) and dsDNA at high affinity. Crystal structure and biochemical analysis confirmed a direct role of the mutations in the acquisition of DNA reactivity and also revealed that these anti-DNA antibodies recognized an unpaired region within DNA duplex. Our study unveils the unique properties of high-affinity anti-DNA antibodies that are generated through antigen-driven affinity maturation in acute phase of SLE.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Antígenos/inmunología , Evolución Clonal/inmunología , Lupus Eritematoso Sistémico/inmunología , Enfermedad Aguda , Secuencia de Aminoácidos , Anticuerpos Antinucleares/química , Antígenos/química , Autoanticuerpos/sangre , ADN/inmunología , Células HEK293 , Humanos , Lupus Eritematoso Sistémico/sangre , Mutación/genética , Tasa de Mutación , Filogenia , Sindecano-1/metabolismoRESUMEN
Laminins regulate diverse cellular functions through interaction with integrins. Two regions of laminins-three laminin globular domains of the α chain (LG1-3) and the carboxyl-terminal tail of the γ chain (γ-tail)-are required for integrin binding, but it remains unclear how the γ-tail contributes to the binding. We determined the crystal structure of the integrin binding fragment of laminin-511, showing that the γ-tail extends to the bottom face of LG1-3. Electron microscopic imaging combined with biochemical analyses showed that integrin binds to the bottom face of LG1-3 with the γ1-tail apposed to the metal ion-dependent adhesion site (MIDAS) of integrin ß1. These findings are consistent with a model in which the γ-tail coordinates the metal ion in the MIDAS through its Glu residue.
Asunto(s)
Integrina alfa6beta1/química , Laminina/química , Sitios de Unión , Integrina alfa6beta1/genética , Integrina alfa6beta1/metabolismo , Iones/química , Laminina/genética , Laminina/metabolismo , Metales/química , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteínas Recombinantes , Relación Estructura-ActividadRESUMEN
Antibody fragments are frequently used as a "crystallization chaperone" to aid structural analysis of complex macromolecules that are otherwise crystallization resistant, but conventional fragment formats have not been designed for this particular application. By fusing an anti-parallel coiled-coil structure derived from the SARAH domain of human Mst1 kinase to the variable region of an antibody, we succeeded in creating a novel chimeric antibody fragment of â¼37 kDa, termed "Fv-clasp," which exhibits excellent crystallization compatibility while maintaining the binding ability of the original IgG molecule. The "clasp" and the engineered disulfide bond at the bottom of the Fv suppressed the internal mobility of the fragment and shielded hydrophobic residues, likely contributing to the high heat stability and the crystallizability of the Fv-clasp. Finally, Fv-clasp antibodies showed superior "chaperoning" activity over conventional Fab fragments, and facilitated the structure determination of an ectodomain fragment of integrin α6ß1.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/química , Proteínas Serina-Treonina Quinasas/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Péptidos y Proteínas de Señalización Intracelular , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Estabilidad ProteicaRESUMEN
The human MutT homolog 1 (hMTH1, human NUDT1) hydrolyzes oxidatively damaged nucleoside triphosphates and is the main enzyme responsible for nucleotide sanitization. hMTH1 recently has received attention as an anticancer target because hMTH1 blockade leads to accumulation of oxidized nucleotides in the cell, resulting in mutations and death of cancer cells. Unlike Escherichia coli MutT, which shows high substrate specificity for 8-oxoguanine nucleotides, hMTH1 has broad substrate specificity for oxidized nucleotides, including 8-oxo-dGTP and 2-oxo-dATP. However, the reason for this broad substrate specificity remains unclear. Here, we determined crystal structures of hMTH1 in complex with 8-oxo-dGTP or 2-oxo-dATP at neutral pH. These structures based on high quality data showed that the base moieties of two substrates are located on the similar but not the same position in the substrate binding pocket and adopt a different hydrogen-bonding pattern, and both triphosphate moieties bind to the hMTH1 Nudix motif (i.e. the hydrolase motif) similarly and align for the hydrolysis reaction. We also performed kinetic assays on the substrate-binding Asp-120 mutants (D120N and D120A), and determined their crystal structures in complex with the substrates. Analyses of bond lengths with high-resolution X-ray data and the relationship between the structure and enzymatic activity revealed that hMTH1 recognizes the different oxidized nucleotides via an exchange of the protonation state at two neighboring aspartate residues (Asp-119 and Asp-120) in its substrate binding pocket. To our knowledge, this mechanism of broad substrate recognition by enzymes has not been reported previously and may have relevance for anticancer drug development strategies targeting hMTH1.