RESUMEN
Simian virus 40 (SV40) provides a model system for the study of eukaryotic DNA replication, in which the viral protein, large T antigen (Tag), marshals human proteins to replicate the viral minichromosome. SV40 replication requires interaction of Tag with the host single-stranded DNA-binding protein, replication protein A (hRPA). The C-terminal domain of the hRPA32 subunit (RPA32C) facilitates initiation of replication, but whether it interacts with Tag is not known. Affinity chromatography and NMR revealed physical interaction between hRPA32C and the Tag origin DNA-binding domain, and a structural model of the complex was determined. Point mutations were then designed to reverse charges in the binding sites, resulting in substantially reduced binding affinity. Corresponding mutations introduced into intact hRPA impaired initiation of replication and primosome activity, implying that this interaction has a critical role in assembly and progression of the SV40 replisome.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Virus 40 de los Simios/crecimiento & desarrollo , Replicación Viral/fisiología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Sitios de Unión , ADN/genética , ADN/metabolismo , Cartilla de ADN/biosíntesis , Cartilla de ADN/genética , Reparación del ADN , Replicación del ADN/fisiología , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/inmunología , Humanos , Modelos Moleculares , Mutación/genética , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Terciaria de Proteína , Proteína de Replicación A , Virus 40 de los Simios/genética , Replicación Viral/efectos de los fármacosRESUMEN
Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's chemokine domain was undertaken. The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors.
Asunto(s)
Quimiocinas CX3C/química , Quimiocinas CX3C/genética , Quimiocinas CX3C/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Arginina/química , Células COS , Calcio/metabolismo , Línea Celular , Células Cultivadas , Quimiocina CX3CL1 , Quimiotaxis , Relación Dosis-Respuesta a Droga , Epítopos , Escherichia coli/metabolismo , Humanos , Cinética , Ligandos , Lisina/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Neuroglía/citología , Fenilalanina/química , Unión Proteica , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia , Factores de Tiempo , TransfecciónRESUMEN
The synthesis of a 93-residue chemokine, lymphotactin, containing eight sites of O-linked glycosylation, was achieved using the technique of native chemical ligation. A single GalNAc residue was incorporated at each glycosylation site using standard Fmoc-chemistry to achieve the first total synthesis of a mucin-type glycoprotein. Using this approach quantities of homogeneous material were obtained for structural and functional analysis.
Asunto(s)
Bioquímica/métodos , Quimiocinas C , Linfocinas/síntesis química , Proteínas de la Membrana , Receptores Acoplados a Proteínas G , Sialoglicoproteínas/síntesis química , Secuencia de Aminoácidos , Células Cultivadas , Glicosilación , Humanos , Riñón/citología , Linfocinas/metabolismo , Linfocinas/farmacología , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación Proteica , Receptores de Superficie Celular/metabolismo , Sialoglicoproteínas/metabolismo , Sialoglicoproteínas/farmacologíaRESUMEN
Fractalkine, or neurotactin, is a chemokine that is present in endothelial cells from several tissues, including brain, liver, and kidney. It is the only member of the CX(3)C class of chemokines. Fractalkine contains a chemokine domain (CDF) attached to a membrane-spanning domain via a mucin-like stalk. However, fractalkine can also be proteolytically cleaved from its membrane-spanning domain to release a freely diffusible form. Fractalkine attracts and immobilizes leukocytes by binding to its receptor, CX(3)CR1. The x-ray crystal structure of CDF has been solved and refined to 2.0 A resolution. The CDF monomers form a dimer through an intermolecular beta-sheet. This interaction is somewhat similar to that seen in other dimeric CC chemokine crystal structures. However, the displacement of the first disulfide in CDF causes the dimer to assume a more compact quaternary structure relative to CC chemokines, which is unique to CX(3)C chemokines. Although fractalkine can bind to heparin in vitro, as shown by comparison of electrostatic surface plots with other chemokines and by heparin chromatography, the role of this property in vivo is not well understood.
Asunto(s)
Quimiocinas CX3C , Quimiocinas CXC/química , Proteínas de la Membrana/química , Quimiocina CX3CL1 , Cristalografía por Rayos X , Modelos Moleculares , Estructura Cuaternaria de ProteínaRESUMEN
The CC chemokine, MCP-1, has been identified as a major chemoattractant for T cells and monocytes, and plays a significant role in the pathology of inflammatory diseases. To identify the regions of MCP-1 that contact its receptor, CCR2, we substituted all surface-exposed residues with alanine. Some residues were also mutated to other amino acids to identify the importance of charge, hydrophobicity, or aromaticity at specific positions. The binding affinity of each mutant for CCR2 was assayed with THP-1 and CCR2-transfected CHL cells. The majority of point mutations had no effect. Residues at the N-terminus of the protein, known to be crucial for signaling, contribute less than a factor of 10 to the binding affinity. However, two clusters of primarily basic residues (R24, K35, K38, K49, and Y13), separated by a 35 A hydrophobic groove, reduced the level of binding by 15-100-fold. A peptide fragment encompassing residues 13-35 recapitulated some of the mutational data derived from the intact protein. It exhibited modest binding as a linear peptide and dramatically improved affinity when the region which adopts a single turn of a 3(10)-helix in the protein, which includes R24, was constrained by a disulfide bond. Additional constraints at the ends of the peptide, corresponding to the disulfide between the first and third cysteines in MCP-1, yielded further improvements in affinity. Together, these data suggest a model in which a large surface area of MCP-1 contacts the receptor, and the accumulation of a number of weak interactions results in the 35 pM affinity observed for the wild-type (WT) protein. The receptor binding site of MCP-1 also is significantly different from the binding sites of RANTES and IL-8, providing insight into the issue of receptor specificity. It was previously shown that the N-terminus of CCR2 is critical for binding MCP-1 [Monteclaro, F. S., and Charo, I. F. (1996) J. Biol. Chem. 271, 19084-92; Monteclaro, F. S., and Charo, I. F. (1997) J. Biol. Chem. 272, 23186-90]. Point mutations of six acidic residues in this region of the receptor were made to test their role in ligand binding. This identified D25 and D27 of the DYDY motif as being important. On the basis of our data, we propose a model in which the receptor N-terminus lies along the hydrophobic groove in an extended fashion, placing the DYDY motif near the basic cluster involving R24 and K49 of MCP-1. This in turn orients the signaling residues (Y13 and the N-terminus) for productive interaction with the receptor.
Asunto(s)
Quimiocina CCL2/química , Quimiocina CCL2/metabolismo , Receptores de Quimiocina/metabolismo , Receptores de Citocinas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular , Quimiocina CCL2/genética , Cricetinae , Cricetulus , Humanos , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Receptores CCR2 , Receptores de Quimiocina/química , Receptores de Citocinas/químicaRESUMEN
Fractalkine, a novel CX3C chemokine, is unusual because of both its membrane-associated structure and its direct role in cell adhesion. We have solved the solution structure of the chemokine domain of fractalkine (residues 1-76) by heteronuclear NMR methods. The 20 lowest energy structures in the ensemble have an average backbone rmsd of 0.43 A, excluding the termini. In contrast to many other chemokines which form homodimers, fractalkine's chemokine module is monomeric. Comparison of the structure to CC and CXC chemokines reveals interesting differences which are likely to be relevant to receptor binding. These include a bulge formed by the CX3C motif, the relative orientation of the N-terminus and 30's loop (residues 30-38), and the conformation of the N-loop (residues 9-19). 15N backbone relaxation experiments indicate that these same regions of the protein are dynamic. We also titrated 15N-labeled protein with a peptide from the N-terminus of the receptor CX3CR1 and confirmed that this region of the receptor contacts the fractalkine chemokine domain. Interestingly, the binding site maps roughly to the regions of greatest flexibility and structural variability. Together, these data provide a first glimpse of how fractalkine interacts with its receptor and should help guide mutagenesis studies to further elucidate the molecular details of binding and signaling through CX3CR1.
Asunto(s)
Quimiocinas CX3C , Quimiocinas CXC/química , Proteínas de la Membrana/química , Fragmentos de Péptidos/química , Receptores de Quimiocina/química , Secuencia de Aminoácidos , Animales , Quimiocina CX3CL1 , Quimiocinas CXC/metabolismo , Simulación por Computador , Cristalografía por Rayos X , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Receptores CXCR3 , Receptores de Quimiocina/metabolismo , Soluciones , TermodinámicaRESUMEN
Injection of lymphotactin (Lptn) into the peritoneum caused an influx of lymphocytes at 24 h. Phenotypic analysis of the cellular influx showed that a large proportion of these cells were T lymphocytes; however, a large number of NK cells was also present. This effect of murine Lptn (mLptn) was specific since the cellular influx was blocked with a mLptn-specific mAb. Similar results were observed when Lptn was injected s.c. and the tissue was analyzed by immunohistochemistry using an anti-CD3epsilon mAb. Microchemotaxis assays confirmed that murine NK cells respond to mLptn, and also showed human NK clones to be similarly responsive to recombinant human Lptn (rhLptn). Immunohistochemical analysis of IL-2-activated murine NK cells and Northern analysis of human NK clones revealed that these cells also produce Lptn, suggesting that a self-regulatory migration mechanism exists in NK cells. Together these data confirm, in vivo, the lymphocyte specificity of Lptn previously observed in vitro and extend its chemotactic effects to the NK cell lineage. We also investigated the functional consequences of truncating the carboxyl terminus of hLptn. This truncated molecule (which is missing the carboxyl-terminal 22 amino acids of hLptn) had no detectable activity on human PBLs. In addition, while hLptn was found to attract murine splenocytes in vitro, the carboxyl-terminal truncated hLptn was again inactive on murine splenocytes. This observation indicates the presence of structural features in the carboxyl terminus of Lptn that are necessary for its biologic activity.