RESUMO
The chemokine stromal cell-derived factor-1 (SDF-1/CXCL12) and its G-protein-coupled receptor (GPCR) CXCR4 play fundamental roles in many physiological processes, and CXCR4 is a drug target for various diseases such as cancer metastasis and human immunodeficiency virus, type 1, infection. However, almost no structural information about the SDF-1-CXCR4 interaction is available, mainly because of the difficulties in expression, purification, and crystallization of CXCR4. In this study, an extensive investigation of the preparation of CXCR4 and optimization of the experimental conditions enables NMR analyses of the interaction between the full-length CXCR4 and SDF-1. We demonstrated that the binding of an extended surface on the SDF-1 beta-sheet, 50-s loop, and N-loop to the CXCR4 extracellular region and that of the SDF-1 N terminus to the CXCR4 transmembrane region, which is critical for G-protein signaling, take place independently by methyl-utilizing transferred cross-saturation experiments along with the usage of the CXCR4-selective antagonist AMD3100. Furthermore, based upon the data, we conclude that the highly dynamic SDF-1 N terminus in the 1st step bound state plays a crucial role in efficiently searching the deeply buried binding pocket in the CXCR4 transmembrane region by the "fly-casting" mechanism. This is the first structural analyses of the interaction between a full-length GPCR and its chemokine, and our methodology would be applicable to other GPCR-ligand systems, for which the structural studies are still challenging.
Assuntos
Quimiocina CXCL12/química , Quimiocina CXCL12/metabolismo , Estrutura Terciária de Proteína , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Quimiocina CXCL12/genética , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Receptores CXCR4/genética , Relação Estrutura-AtividadeRESUMO
Human thrombopoietin (hTPO) is a primary hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production. The non-glycosylated form of 1-163 residues of hTPO (hTPO163 ) including the N-terminal active site domain (1-153 residues) is a candidate for treating thrombocytopenia. However, the autoantigenicity level of hTPO163 is higher than that of the full-length glycosylated hTPO (ghTPO332 ). In order to clarify the structural and physicochemical properties of hTPO163 , circular dichroism (CD) and differential scanning calorimetry (DSC) analyses were performed. CD analysis indicated that hTPO163 undergoes an induced-fit conformational change (+19.0% for helix and -16.7% for ß-strand) upon binding to the neutralizing antibody TN1 in a manner similar to the coupled folding and binding mechanism. Moreover, DSC analysis showed that the thermal transition process of hTPO163 is a multistate transition; hTPO163 is thermally stabilized upon receptor (c-Mpl) binding, as indicated with raising the midpoint (Tm ) temperature of the transition by at least +9.5 K. The conformational variability and stability of hTPO163 indicate that hTPO163 exists as a molten globule under native conditions, which may enable the induced-fit conformational change according to the type of ligands (antibodies and receptor). Additionally, CD and computational analyses indicated that the C-terminal domain (154-332 residues) and glycosylation assists the folding of the N-terminal domain. These observations suggest that the antibody affinity and autoantigenicity of hTPO163 might be reduced, if the conformational variability of hTPO163 is restricted by mutation and/or by the addition of C-terminal domain with glycosylation to keep its conformation suitable for the c-Mpl recognition.
Assuntos
Trombopoetina/química , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Humanos , Conformação Proteica , Dobramento de Proteína , Trombopoetina/imunologiaRESUMO
The aorta-gonad-mesonephros (AGM) region is involved in the generation and maintenance of the first definitive hematopoietic stem cells (HSCs). A mouse AGM-derived cell line, AGM-S3, was shown to support the development of HSCs. To elucidate the molecular mechanisms regulating early hematopoiesis, we obtained subclones from AGM-S3, one of which was hematopoiesis supportive (S3-A9) and the other one of which was non-supportive (S3-A7), and we analyzed their gene expression profiles by gene chip analysis. In the present study, we found that Glypican-1 (GPC1) was highly expressed in the supportive subclone AGM-S3-A9. Over-expression of GPC1 in non-supportive cells led to the proliferation of progenitor cells in human cord blood when cocultured with the transfected-stromal cells. Thus, GPC1 may have an important role in the establishment of a microenvironment that supports early events in hematopoiesis.
Assuntos
Aorta/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Glipicanas/genética , Gônadas/metabolismo , Hematopoese/genética , Mesonefro/metabolismo , Animais , Aorta/citologia , Aorta/embriologia , Linhagem Celular , Proliferação de Células , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Perfilação da Expressão Gênica , Glipicanas/fisiologia , Gônadas/citologia , Gônadas/embriologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Mesonefro/citologia , Camundongos , Células Estromais/citologia , Células Estromais/metabolismo , TransfecçãoRESUMO
Human thrombopoietin (hTPO) primarily stimulates megakaryocytopoiesis and platelet production and is neutralized by the mouse TN1 antibody. The thermodynamic characteristics of TN1 antibody-hTPO complexation were analyzed by isothermal titration calorimetry (ITC) using an antigen-binding fragment (Fab) derived from the TN1 antibody (TN1-Fab). To clarify the mechanism by which hTPO is recognized by TN1-Fab the conformation of free TN1-Fab was determined to a resolution of 2.0 Å using X-ray crystallography and compared with the hTPO-bound form of TN1-Fab determined by a previous study. This structural comparison revealed that the conformation of TN1-Fab does not substantially change after hTPO binding and a set of 15 water molecules is released from the antigen-binding site (paratope) of TN1-Fab upon hTPO complexation. Interestingly, the heat capacity change (ΔCp) measured by ITC (-1.52 ± 0.05 kJ mol(-1) K(-1) ) differed significantly from calculations based upon the X-ray structure data of the hTPO-bound and unbound forms of TN1-Fab (-1.02 â¼ 0.25 kJ mol(-1) K(-1) ) suggesting that hTPO undergoes an induced-fit conformational change combined with significant desolvation upon TN1-Fab binding. The results shed light on the structural biology associated with neutralizing antibody recognition.
Assuntos
Anticorpos Monoclonais Murinos/química , Fragmentos Fab das Imunoglobulinas/química , Trombopoetina/química , Animais , Cristalografia por Raios X , Humanos , Camundongos , Estrutura Quaternária de Proteína , TermodinâmicaRESUMO
Previously, we have reported that cationized-proteins covalently modified with polyethylenimine (PEI) (direct PEI-cationization) efficiently enter cells and function in the cytosol [Futami et al. (2005) J. Biosci. Bioeng. 99, 95-103]. However, it may be more convenient if a protein could be delivered into cells just by mixing the protein with a PEI-cationized carrier protein having a specific affinity (indirect PEI-cationization). Thus, we prepared PEI-cationized avidin (PEI-avidin), streptavidin (PEI-streptavidin), and protein G (PEI-protein G), and examined whether they could deliver biotinylated proteins and antibodies into living cells. PEI-avidin (and/or PEI-streptavidin) carried biotinylated GFPs into various mammalian cells very efficiently. A GFP variant containing a nuclear localization signal was found to arrive even in the nucleus. The addition of a biotinylated RNase A derivative mixed with PEI-streptavidin to a culture medium of 3T3-SV-40 cells resulted in remarkable cell growth inhibition, suggesting that the biotinylated RNase A derivative entered cells and digested intracellular RNA molecules. Furthermore, the addition of a fluorescein-labeled anti-S100C (beta-actin binding protein) antibody mixed with PEI-protein G to human fibroblasts resulted in the appearance of a fluorescence image of actin-like filamentous structures in the cells. These results indicate that indirect PEI-cationization using non-covalent interaction is as effective as the direct PEI-cationization for the transduction of proteins into living cells and for expression of their functions in the cytosol. Thus, PEI-cationized proteins having a specific affinity for certain molecules such as PEI-streptavidin, PEI-avidin and PEI-protein G are concluded to be widely applicable protein transduction carrier molecules.
Assuntos
Biotecnologia/métodos , Polietilenoimina/metabolismo , Transporte Proteico , Animais , Avidina , Células 3T3 BALB , Biotinilação , Proteínas de Transporte/metabolismo , Cátions/química , Cátions/metabolismo , Morte Celular , Fibroblastos , Humanos , Camundongos , Microscopia Confocal , Proteínas do Tecido Nervoso , Polietilenoimina/química , Ribonuclease Pancreático , EstreptavidinaRESUMO
The granulocyte-colony stimulating factor (GCSF) receptor receives signals for regulating the maturation, proliferation and differentiation of the precursor cells of neutrophilic granulocytes. The signalling complex composed of two GCSFs (GCSF, 19 kDa) and two GCSF receptors (GCSFR, 34 kDa) consisting of an Ig-like domain and a cytokine-receptor homologous (CRH) domain was crystallized. A crystal of the complex was grown in 1.0 M sodium formate and 0.1 M sodium acetate pH 4.6 and belongs to space group P4(1)2(1)2 (or its enantiomorph P4(3)2(1)2), with unit-cell parameters a = b = 110.1, c = 331.8 A. Unfortunately, this crystal form did not diffract beyond 5 A resolution. Since the heterogeneity of GCSF receptor appeared to prevent the growth of good-quality crystals, the GCSF receptor was fractionated by anion-exchange chromatography. Crystals of the GCSF-fractionated GCSF receptor complex were grown as a new crystal form in 0.2 M ammonium phosphate. This new crystal form diffracted to beyond 3.0 A resolution and belonged to space group P3(1)21 (or its enantiomorph P3(2)21), with unit-cell parameters a = b = 134.8, c = 105.7 A.
Assuntos
Fator Estimulador de Colônias de Granulócitos/química , Fator Estimulador de Colônias de Granulócitos/metabolismo , Substâncias Macromoleculares/química , Receptores de Fator Estimulador de Colônias de Granulócitos/química , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Cromatografia por Troca Iônica , Cristalização , Eletroforese em Gel de Poliacrilamida , Humanos , Ligantes , Proteínas Recombinantes/química , Difração de Raios XRESUMO
A requirement for advancing antibody-based medicine is the development of proteins that can bind with high affinity to a specific epitope related to a critical protein activity site. As a part of generating such proteins, we have succeeded in creating a binding protein without changing epitope by complementarity-determining region 3 (CDR3) grafting (Inoue et al., Affinity transfer to a human protein by CDR3 grafting of camelid VHH. Protein Sci. 20, 1971-1981). However, the affinity of the target-binding protein was low. In this manuscript, the affinity maturation of a target-binding protein was examined using CDR3-grafted camelid single domain antibody (VHH) as a model protein. Several amino acids in the CDR1 and CDR2 regions of VHH were mutated to tyrosines and/or serines and screened for affinity-matured proteins by using in silico analysis. The mutation of two amino acids in the CDR2 region to arginine and/or aspartic acid increased the affinity by decreasing the dissociation rate. The affinity of designed mutant increased by â¼20-fold over that of the original protein. In the present study, candidate mutants were narrowed down using in silico screening and computational modelling, thus avoiding much in vitro analytical effort. Therefore, the method used in this study is expected to be one of the useful for promoting affinity maturation of antibodies.
Assuntos
Anticorpos de Domínio Único/química , Sequência de Aminoácidos , Animais , Camelídeos Americanos , Simulação por Computador , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Anticorpos de Domínio Único/genética , Ressonância de Plasmônio de SuperfícieRESUMO
VHH is the binding domain of the IgG heavy chain. Some VHHs have an extremely long CDR3 that contributes to antigen binding. We studied the antigen binding ability of CDR3 by grafting a CDR3 from an antigen-binding VHH onto a nonbinding VHH. cAb-CA05-(1RI8), the CDR3-grafted VHH, had an antigen-binding ability. To find a human scaffold protein acceptable for VHH CDR3 grafting, we focused on the conserved structure of VHH, especially the N-terminal and C-terminal amino acid residues of the CDR3 loop and the Cys residue of CDR1. Human origin protein structures with the same orientation were searched in PDB and ubiquitin was selected. Ubi-(1RI8), the CDR3-grafted ubiquitin, had antigen-binding ability, though the affinity was relatively low compared to cAb-CA05-(1RI8). The thermodynamic parameters of Ubi-(1RI8) binding to HEWL were different from cAb-CA05-(1RI8). Hydrogen-deuterium exchange experiments showed decreased stability around the CDR3 grafting region of Ubi-(1RI8), which might explain the decreased antigen-binding ability and the differences in thermodynamic properties. We concluded that the orientation of the CDR3 sequence of Ubi-(1RI8) could not be reconstructed correctly.
Assuntos
Camelídeos Americanos/imunologia , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/imunologia , Ubiquitina/química , Ubiquitina/imunologia , Sequência de Aminoácidos , Animais , Escherichia coli/genética , Expressão Gênica , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Alinhamento de Sequência , Ubiquitina/genéticaRESUMO
The Wnt canonical signaling pathway is essential for the early development of eukaryotic organisms and plays a key role in cell proliferation, differentiation, and oncogenesis. Moreover, the Wnt canonical signaling pathway contributes to the self-renewal of mouse hematopoietic stem cells (HSCs). Here, we demonstrate artificial activation of the Wnt canonical signaling pathway by beta-catenin protein transduction. Constitutively active beta-catenin protein was introduced into human embryonic kidney HEK-293 cells using a polyethylenimine (PEI) cationization method, or with the BioPORTER protein transduction reagent. We have previously shown that modification with PEI effectively causes proteins to be internalized by living mammalian cells. PEI-cationized, constitutively active beta-catenin protein was added to HEK-293 cells, and induction of several Wnt/beta-catenin target genes was detected by real-time PCR. However, using BioPORTER to introduce active beta-catenin did not activate the Wnt canonical signaling pathway. Introduction of eGFPNuc (enhanced green fluorescent protein variant containing a nuclear localization signal) into HEK-293 cells using the BioPORTER reagent caused significant cell death, as determined by propidium iodide staining. In contrast, the PEI-modified eGFPNuc did not impair survival of HEK-293 cells. These results indicate that the Wnt canonical signaling pathway could be successfully activated by transduction of PEI-cationized active beta-catenin, and the PEI-cationization method is an effective and safe technology for protein transduction into mammalian cells.
Assuntos
Portadores de Fármacos/química , Rim/fisiologia , Polietilenoimina/química , Transdução de Sinais/fisiologia , Transdução Genética/métodos , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Cátions , Linhagem Celular , Transfecção/métodos , beta Catenina/genéticaRESUMO
A crystal structure of the signaling complex between human granulocyte colony-stimulating factor (GCSF) and a ligand binding region of GCSF receptor (GCSF-R), has been determined to 2.8 A resolution. The GCSF:GCSF-R complex formed a 2:2 stoichiometry by means of a cross-over interaction between the Ig-like domains of GCSF-R and GCSF. The conformation of the complex is quite different from that between human GCSF and the cytokine receptor homologous domain of mouse GCSF-R, but similar to that of the IL-6/gp130 signaling complex. The Ig-like domain cross-over structure necessary for GCSF-R activation is consistent with previously reported thermodynamic and mutational analyses.
Assuntos
Fator Estimulador de Colônias de Granulócitos/química , Fator Estimulador de Colônias de Granulócitos/metabolismo , Receptores de Fator Estimulador de Colônias de Granulócitos/química , Receptores de Fator Estimulador de Colônias de Granulócitos/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Cristalografia por Raios X , Receptor gp130 de Citocina/química , Receptor gp130 de Citocina/metabolismo , Dimerização , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Proteínas Recombinantes , Alinhamento de Sequência , Homologia Estrutural de ProteínaRESUMO
In order to perform biochemical and pharmacological characterization of CXCR1, we designed several CXCR1 constructs. All constructs, including a CXCR1-G(i2)alpha fusion protein, were produced in insect cells after infection with recombinant baculovirus. The recombinant receptors exhibited specific high-affinity binding of (125)I-labelled interleukin-8, and Scatchard transformation of the binding data indicated the presence of a population of single homogenous binding sites. Furthermore, the pharmacological profiles for the different CXCR1 constructs produced in the baculovirus-infected insect cells were almost identical to those reported for CXCR1 on human neutrophils. Interestingly, when the CXCR1 constructs were coproduced with G(i2) protein as a result of coinfection with baculoviruses encoding the G(i2)alpha-, the beta- and the gamma- subunits, the B(max) values were significantly increased. Hence, the level of FlagCXCR1Bio, after coproduction with G(i2) protein, was found to be almost 10 times higher than that of the FlagCXCR1Bio alone. However, no differences in the K(i) values were observed of the receptor constructs produced either after single infection or coinfection of insect cells. The addition of guanyl-5'-yl imidodiphosphate resulted in a dramatic reduction of the number of binding sites; however, the K(i) values remained unchanged, indicating coupling of the receptor to the guanine nucleotide-binding protein.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Interleucina-8A/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Baculoviridae/genética , Linhagem Celular , Subunidade alfa Gi2 de Proteína de Ligação ao GTP , Glicosilação , Guanilil Imidodifosfato/farmacologia , Humanos , Receptores de Interleucina-8A/análise , Receptores de Interleucina-8A/imunologia , SpodopteraRESUMO
Thrombopoietin (TPO) is a cytokine which primarily stimulates megakaryocytopoiesis and thrombopoiesis. The functional domain of TPO (TPO(163)) consisting of the N-terminal 163 amino acids was prepared and crystallized. Since the crystallization of TPO(163) was unsuccessful using the standard screening methods, a Fab fragment derived from a neutralizing monoclonal antibody was used for crystallization. It was found that the TPO(163)-Fab complex crystallized reproducibly in 0.1 M potassium phosphate buffer pH 6.0 containing 20-25% polyethylene glycol 4000. Thin crystals (0.2 x 0.2 x 0.02 mm) grew in two space groups: P2(1), with unit-cell parameters a = 133.20, b = 46.71, c = 191.47 A, beta = 90.24 degrees, and C2, with unit-cell parameters a = 131.71, b = 46.48, c = 184.63 A, beta = 90.42 degrees. The results of a molecular-replacement analysis indicate that the Fab molecules interact with each other and provide a suitable interface for crystallization.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Trombopoetina/química , Trombopoetina/imunologia , Cristalização , Humanos , Substâncias Macromoleculares , Ligação Proteica , Estrutura Terciária de Proteína , Difração de Raios X/métodosRESUMO
The cytokine thrombopoietin (TPO), the ligand for the hematopoietic receptor c-Mpl, acts as a primary regulator of megakaryocytopoiesis and platelet production. We have determined the crystal structure of the receptor-binding domain of human TPO (hTPO(163)) to a 2.5-A resolution by complexation with a neutralizing Fab fragment. The backbone structure of hTPO(163) has an antiparallel four-helix bundle fold. The neutralizing Fab mainly recognizes the C-D crossover loop containing the species invariant residue Q111. Titration calorimetric experiments show that hTPO(163) interacts with soluble c-Mpl containing the extracellular cytokine receptor homology domains with 1:2 stoichiometry with the binding constants of 3.3 x 10(9) M(-1) and 1.1 x 10(6) M(-1). The presence of the neutralizing Fab did not inhibit binding of hTPO(163) to soluble c-Mpl fragments, but the lower-affinity binding disappeared. Together with prior genetic data, these define the structure-function relationships in TPO and the activation scheme of c-Mpl.
Assuntos
Fragmentos Fab das Imunoglobulinas/imunologia , Trombopoetina/química , Trombopoetina/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Testes de Neutralização , Ligação Proteica , Conformação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Trombopoetina , Solubilidade , Relação Estrutura-Atividade , Especificidade por Substrato , Trombopoetina/imunologiaRESUMO
The granulocyte colony-stimulating factor receptor (GCSFR), containing the Ig-like domain (Ig) and cytokine receptor homologous region (CRH), was prepared as a preformed dimer (Ig-CRH-Fc)(2) after fusion to the mouse Fc region via an eight-residue linker (approximately 55 A). Monomer Ig-CRH was also prepared after the Fc region was removed from (Ig-CRH-Fc)(2). GCSF binding to Ig-CRH and (Ig-CRH-Fc)(2) was investigated using light scattering and isothermal titration calorimetry. The average molecular mass determined by light scattering showed that both Ig-CRH and (Ig-CRH-Fc)(2) formed a 2:2 dimer with GCSF. Moreover, isothermal titration calorimetry showed that the thermodynamic parameters upon binding of GCSF to Ig-CRH and (Ig-CRH-Fc)(2) were comparable, suggesting a similar binding stoichiometry and interface [including similar buried surface area (5700-6000 A(2))] despite the presence of the eight-residue linker. The buried surface area is much larger than that calculated from our previous report of the crystal structure of the GCSF-CRH complex [Aritomi, M., et al. (1999) Nature 401, 713-717], suggesting a substantial contribution of the Ig domain to GCSF binding. The data also indicate that the distance (55 A) between two CRH domains in the 2:2 complex is much shorter than in our previous model (approximately 90 A) predicted from the same crystal structure of the GCSF-CRH complex.