ABSTRACT
The objectives of this study were to evaluate the relative binding and potencies of three inhibitors of vascular endothelial growth factor A (VEGF), used to treat neovascular age-related macular degeneration, and assess their relevance in the context of clinical outcome. Ranibizumab is a 48 kDa antigen binding fragment, which lacks a fragment crystallizable (Fc) region and is rapidly cleared from systemic circulation. Aflibercept, a 110 kDa fusion protein, and bevacizumab, a 150 kDa monoclonal antibody, each contain an Fc region. Binding affinities were determined using Biacore analysis. Competitive binding by sedimentation velocity analytical ultracentrifugation (SV-AUC) was used to support the binding affinities determined by Biacore of ranibizumab and aflibercept to VEGF. A bovine retinal microvascular endothelial cell (BREC) proliferation assay was used to measure potency. Biacore measurements were format dependent, especially for aflibercept, suggesting that biologically relevant, true affinities of recombinant VEGF (rhVEGF) and its inhibitors are yet to be determined. Despite this assay format dependency, ranibizumab appeared to be a very tight VEGF binder in all three formats. The results are also very comparable to those reported previously.1-3 At equivalent molar ratios, ranibizumab was able to displace aflibercept from preformed aflibercept/VEGF complexes in solution as assessed by SV-AUC, whereas aflibercept was not able to significantly displace ranibizumab from preformed ranibizumab/VEGF complexes. Ranibizumab, aflibercept, and bevacizumab showed dose-dependent inhibition of BREC proliferation induced by 6 ng/mL VEGF, with average IC50 values of 0.088 ± 0.032, 0.090 ± 0.009, and 0.500 ± 0.091 nM, respectively. Similar results were obtained with 3 ng/mL VEGF. In summary Biacore studies and SV-AUC solution studies show that aflibercept does not bind with higher affinity than ranibizumab to VEGF as recently reported,4 and both inhibitors appeared to be equipotent with respect to their ability to inhibit VEGF function.
Subject(s)
Antibodies, Monoclonal, Humanized/metabolism , Receptors, Vascular Endothelial Growth Factor/metabolism , Recombinant Fusion Proteins/metabolism , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/metabolism , Animals , Bevacizumab , Cattle , Cell Line , Cell Proliferation/drug effects , Endothelial Cells/cytology , Endothelial Cells/drug effects , Protein Binding , Ranibizumab , Ultracentrifugation , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
The ability to leverage antibodies to agonize disease relevant biological pathways has tremendous potential for clinical investigation. Yet while antibodies have been successful as antagonists, immune mediators, and targeting agents, they are not readily effective at recapitulating the biology of natural ligands. Among the important determinants of antibody agonist activity is the geometry of target receptor engagement. Here, we describe an engineering approach inspired by a naturally occurring Fab-Fab homotypic interaction that constrains IgG in a unique i-shaped conformation. i-shaped antibody (iAb) engineering enables potent intrinsic agonism of five tumor necrosis factor receptor superfamily (TNFRSF) targets. When applied to bispecific antibodies against the heterodimeric IL-2 receptor pair, constrained bispecific IgG formats recapitulate IL-2 agonist activity. iAb engineering provides a tool to tune agonist antibody function and this work provides a framework for the development of intrinsic antibody agonists with the potential for generalization across broad receptor classes.
Subject(s)
Antibodies, Bispecific , Receptors, Tumor Necrosis Factor , Immunoglobulin G/genetics , Protein EngineeringABSTRACT
We describe a general Biacore method for measuring equilibrium binding affinities and stoichiometries for interactions between unmodified proteins and their unmodified ligands free in solution. Mixtures of protein and ligand are preequilibrated at different ratios in solution and then analyzed by Biacore using a sensor chip surface that detects only unbound analyte. Performing the Biacore analysis under mass transport limited conditions allows the concentration of unbound analyte to be determined from the initial velocity of binding. Plots of initial velocity versus the concentration of the varied binding partner are fitted to a quadratic binding equation to give the affinity and stoichiometry of binding. We demonstrate the method using soluble Her2 extracellular domain binding to monovalent, bivalent, and trivalent forms of an anti-Her2 antibody. The affinity we measured agrees with that obtained from conventional Biacore kinetic analysis, and the stoichiometries for the resulting 1:1, 1:2, and 1:3 complexes were confirmed by gel filtration with in-line light scattering. The method is applicable over an affinity range of approximately 100 pM to 1 µM and is particularly useful when there is concern that covalently modifying one or the other binding partner might affect its binding properties or where multivalency might otherwise complicate a quantitative analysis of binding.
Subject(s)
Chromatography, Gel/methods , Proteins/analysis , Receptor, ErbB-2/metabolism , Surface Plasmon Resonance/methods , Animals , Cricetinae , Cricetulus , Humans , Kinetics , Protein Binding , Proteins/chemistry , Receptor, ErbB-2/analysisABSTRACT
We examine the relationship between binding affinity and interface size for reversible protein-protein interactions (PPIs), using cytokines from the tumor necrosis factor (TNF) superfamily and their receptors as a test case. Using surface plasmon resonance, we measured single-site binding affinities for binding of the large receptor TNFR1 to its ligands TNFα (K(D) = 1.4 ± 0.4 nM) and lymphotoxin-α (K(D) = 50 ± 10 nM), and also for binding of the small receptor Fn14 to TWEAK (K(D) = 70 ± 10 nM). We additionally assembled data for all other TNF-TNFR family complexes for which reliable single-site binding affinities have been reported. We used these values to calculate the binding efficiencies, defined as binding energy per square angstrom of surface area buried at the contact interface, for nine of these complexes for which cocrystal structures are available, and compared the results to those for a set of 144 protein-protein complexes with published affinities. The results show that the most efficient PPI complexes generate ~20 cal mol(-1) Å(-2) of binding energy. A minimal contact area of ~500 Å(2) is required for a stable complex, required to generate sufficient interaction energy to pay the entropic cost of colocalizing two proteins from 1 M solution. The most compact and efficient TNF-TNFR complex was the BAFF-BR3 complex, which achieved ~80% of the maximal achievable binding efficiency. Other small receptors also gave high binding efficiencies, while the larger receptors generated only 44-49% of this limit despite interacting primarily through just a single small domain. The results provide new insight into how much binding energy can be generated by a PPI interface of a given size, and establish a quantitative method for predicting how large a natural or engineered contact interface must be to achieve a given level of binding affinity.
Subject(s)
Lymphotoxin-alpha/metabolism , Protein Binding , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Necrosis Factors/metabolism , Cytokine TWEAK , Kinetics , Ligands , Receptors, Tumor Necrosis Factor/metabolism , Surface Plasmon Resonance , TWEAK ReceptorABSTRACT
Analytical ultracentrifugation (AUC) is a critical analytical tool supporting the development and manufacture of protein therapeutics. AUC is routinely used as an assay orthogonal to size exclusion chromatography for aggregate quantitation. This article distills the experimental and analysis procedures used by the authors for sedimentation velocity AUC into a series of best-practices considerations. The goal of this distillation is to help harmonize aggregate quantitation approaches across the biopharmaceutical industry. We review key considerations for sample and instrument suitability, experimental design, and data analysis best practices and conversely, highlight potential pitfalls to accurate aggregate analysis. Our goal is to provide experienced users benchmarks against which they can standardize their analyses and to provide guidance for new AUC analysts that will aid them to become proficient in this fundamental technique.
Subject(s)
Antibodies, Monoclonal , Antibodies, Monoclonal/chemistry , Chromatography, Gel , Ultracentrifugation/methodsABSTRACT
The port delivery system with ranibizumab (PDS) is an investigational long-acting drug delivery system for the continuous release of ranibizumab, an anti-VEGF biologic, in the vitreous humor. The efficacy of the PDS implant relies on the maintenance of long-term drug stability under physiological conditions. Herein, the long-term stability of three anti-VEGF biologics - ranibizumab, bevacizumab and aflibercept - was investigated in phosphate buffered saline (PBS) at 37 °C for several months. Comparison of stability profiles shows that bevacizumab and aflibercept are increasingly prone to aggregation whereas ranibizumab undergoes minimal aggregation. Ranibizumab also shows the smallest loss in antigen binding capacity after long-term incubation in PBS. Even though the aggregated forms of bevacizumab and aflibercept bind to VEGF, the consequences of aggregation on immunogenicity, implant function and efficacy are unknown. These results highlight the importance of maintaining long-term drug stability under physiologically relevant conditions which is necessary for achieving efficacy with an in vivo continuous drug delivery device such as the PDS implant.
Subject(s)
Biological Products , Vascular Endothelial Growth Factor A , Angiogenesis Inhibitors , Bevacizumab , Intravitreal Injections , Ranibizumab , Recombinant Fusion ProteinsABSTRACT
Inositol-Requiring Enzyme 1 (IRE1) is an essential component of the Unfolded Protein Response. IRE1 spans the endoplasmic reticulum membrane, comprising a sensory lumenal domain, and tandem kinase and endoribonuclease (RNase) cytoplasmic domains. Excess unfolded proteins in the ER lumen induce dimerization and oligomerization of IRE1, triggering kinase trans-autophosphorylation and RNase activation. Known ATP-competitive small-molecule IRE1 kinase inhibitors either allosterically disrupt or stabilize the active dimeric unit, accordingly inhibiting or stimulating RNase activity. Previous allosteric RNase activators display poor selectivity and/or weak cellular activity. In this study, we describe a class of ATP-competitive RNase activators possessing high selectivity and strong cellular activity. This class of activators binds IRE1 in the kinase front pocket, leading to a distinct conformation of the activation loop. Our findings reveal exquisitely precise interdomain regulation within IRE1, advancing the mechanistic understanding of this important enzyme and its investigation as a potential small-molecule therapeutic target.
Subject(s)
Adenosine Triphosphate/metabolism , Endoribonucleases/metabolism , Protein Serine-Threonine Kinases/metabolism , Ribonucleases/metabolism , Adenosine Triphosphate/chemistry , Allosteric Site/drug effects , Crystallography, X-Ray , Endoplasmic Reticulum/metabolism , Endoribonucleases/chemistry , Gene Knockout Techniques , Humans , Ligands , Models, Molecular , Phosphorylation , Protein Conformation , Protein Folding , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Multimerization , Protein Serine-Threonine Kinases/chemistry , Ribonucleases/chemistry , Unfolded Protein ResponseABSTRACT
Enhancement of antigen-specific T cell immunity has shown significant therapeutic benefit in infectious diseases and cancer. Hematopoietic progenitor kinase-1 (HPK1) is a negative-feedback regulator of T cell receptor signaling, which dampens T cell proliferation and effector function. A recent report showed that a catalytic dead mutant of HPK1 phenocopies augmented T cell responses observed in HPK1-knockout mice, indicating that kinase activity is critical for function. We evaluated active and inactive mutants and determined crystal structures of HPK1 kinase domain (HPK1-KD) in apo and ligand bound forms. In all structures HPK1-KD displays a rare domain-swapped dimer, in which the activation segment comprises a well-conserved dimer interface. Biophysical measurements show formation of dimer in solution. The activation segment adopts an α-helical structure which exhibits distinct orientations in active and inactive states. This face-to-face configuration suggests that the domain-swapped dimer may possess alternative selectivity for certain substrates of HPK1 under relevant cellular context.
Subject(s)
Catalytic Domain , Protein Multimerization , Protein Serine-Threonine Kinases/chemistry , Animals , Humans , Molecular Dynamics Simulation , Protein Binding , Protein Serine-Threonine Kinases/metabolism , Sf9 Cells , SpodopteraABSTRACT
Subvisible particle formation in monoclonal antibody drug product resulting from mixing and filling operations represents a significant processing risk that can lead to filter fouling and thereby lead to process delays or failures. Several previous studies from our lab and others demonstrated the formation of subvisible particulates in mAb formulations resulting from mixing operations using some bottom-mounted mixers or stirrer bars. It was hypothesized that the stress (e.g., shear/cavitation) derived from tight clearance and/or close contact between the impeller and shaft was responsible for protein subvisible particulate generation. These studies, however, could not distinguish between the two surfaces without contact (tight clearance) or between two contacting surfaces (close contact). In the present study we expand on those findings and utilize small-scale mixing models that are able to, for the first time, distinguish between tight clearances and tight contact. In this study we evaluated different mixer types including a top-mounted mixer, several impeller-based bottom-mounted mixers, and a rotary piston pump. The impact of tight clearance/close contact on subvisible particle formation in at-scale mixing platforms was demonstrated in the gap between the impeller and drive unit as well as between the piston and the housing of the pump. Furthermore, small-scale mixing models based on different designs of magnetic stir bars that mimic the tight clearance/close contact of the manufacturing-scale mixers also induced subvisible particles in mAb formulations. Additional small-scale models that feature tight clearance but no close contact (grinding) suggested that it is the repeated grinding/contacting of the moving parts and not the presence of tight clearance in the processing equipment that is the root cause of protein subvisible particulate formation. When multiple mAbs, Fabs (fragment antigen binding), or non-antibody related proteins were mixed in the small-scale mixing model, for molecules investigated, it was observed that mAbs and Fabs appear to be more susceptible to particle formation than non-antibody-related proteins. In the grinding zone, mAb/Fab molecules aggregated into insoluble particles with neither detectable soluble aggregates nor fragmented species. This investigation represents a step closer to the understanding of the underlying stress mechanism leading to mAb subvisible particulate formation as the result of drug product processing.LAY ABSTRACT: Mixing and fill finish are important unit operations in drug product manufacturing for compounding (dilution, pooling, homogenization, etc.) and filling into primary packaging containers (vials, pre-filled syringes, etc.), respectively. The current trend in adopting bottom-mounted mixers as well as low fill-volume filling systems has raised concerns about their impact on drug product quality and process performance. However, investigations into the effects of their use for biopharmaceutical products, particularly monoclonal antibody formulations, are rarely published. The purpose of this study is three-fold: (1) to revisit the impact of bottom-mounted mixer design on monoclonal antibody subvisible particle formation; (2) to identify the root cause for subvisible particle formation; and (3) to fully utilize available particle analysis tools to demonstrate the correlation between particle count in the solution and filter fouling during sterile filtration. The outcomes of this study will benefit scientists and engineers who develop biologic product manufacturing processes by providing a better understanding of drug product process challenges.
Subject(s)
Antibodies, Monoclonal/chemistry , Immunoglobulin Fab Fragments/chemistry , Pharmaceutical Preparations/chemistry , Technology, Pharmaceutical/methods , Chromatography, Gel , Chromatography, High Pressure Liquid , Drug Compounding , Equipment Design , Protein Aggregates , Stress, Mechanical , Technology, Pharmaceutical/instrumentation , UltracentrifugationABSTRACT
A microtiter plate assay measuring the binding of cells expressing integrins alpha4beta1 or alpha4beta7 to VCAM-1 and MAdCAM-1, expressed as Ig fusion proteins, was used to explore the interplay between the variables of integrin beta-chain, identity and density of ligand, and identity and concentration of activating cations. Both Mn2+ and Mg2+ supported binding of either integrin to either ligand. Ca2+ supported only the binding of alpha4beta1 to VCAM-Ig. Cation concentrations required for half-maximal binding (EC50) ranged from 0.8-280 microM for Mn2+ and 0.8-30 mM for Mg2+, being thus 2-3 logs lower for Mn2+ compared to Mg2+ independent of ligand. EC50 values for binding of alpha4beta1 to VCAM-Ig were 30-45-fold lower compared to MAdCAM-Ig, while alpha4beta7 showed an opposite 3-15-fold selectivity for MAdCAM-Ig over VCAM-Ig. The density of ligand required for adhesion via alpha4beta1 was markedly lower with Mn2+ versus Mg2+, and with VCAM-Ig versus MAdCAM-Ig. These results were interpreted in terms of a coupled equilibrium model, in which binding of activating metal ions and of integrin ligands each stabilizes activated integrin. We conclude that Mn2+ and Mg2+ bind to common regulatory sites with different affinities, producing similar activated states of the integrin. The resulting activated alpha4beta1 binds more strongly to VCAM-Ig versus MAdCAM-Ig by 30-45-fold, while similarly activated alpha4beta7 binds more strongly to MAdCAM-Ig versus VCAM-Ig by 3-15-fold. Inhibition studies showed that Ca2+ also binds to regulatory sites on both integrins. However, the Ca2+-activated state of alpha4beta1 is distinct from that achieved by Mn2+ and Mg2+, possessing increased selectivity for binding to VCAM-1 versus MAdCAM-1.
Subject(s)
Calcium/metabolism , Immunoglobulins/metabolism , Integrin alpha4beta1/metabolism , Integrins/metabolism , Mucoproteins/metabolism , Vascular Cell Adhesion Molecule-1/metabolism , Antibodies, Monoclonal , Binding Sites , Calcium/pharmacology , Calcium/physiology , Cell Adhesion/physiology , Cell Adhesion Molecules , Humans , Immunoglobulins/immunology , In Vitro Techniques , Integrin alpha4beta1/agonists , Integrin alpha4beta1/physiology , Integrins/agonists , Integrins/physiology , Jurkat Cells , Lymphoma, B-Cell , Magnesium/metabolism , Magnesium/pharmacology , Magnesium/physiology , Manganese/metabolism , Manganese/pharmacology , Manganese/physiology , Mucoproteins/immunology , Tumor Cells, Cultured , Vascular Cell Adhesion Molecule-1/immunologyABSTRACT
TWEAK (TNF homologue with weak apoptosis-inducing activity) and Fn14 (fibroblast growth factor-inducible protein 14) are members of the tumor necrosis factor (TNF) ligand and receptor super-families. Having observed that Xenopus Fn14 cross-reacts with human TWEAK, despite its relatively low sequence homology to human Fn14, we examined the conservation in tertiary fold and binding interfaces between the two species. Our results, combining NMR solution structure determination, binding assays, extensive site-directed mutagenesis and molecular modeling, reveal that, in addition to the known and previously characterized ß-hairpin motif, the helix-loop-helix motif makes an essential contribution to the receptor/ligand binding interface. We further discuss the insight provided by the structural analyses regarding how the cysteine-rich domains of the TNF receptor super-family may have evolved over time. DATABASE: Structural data are available in the Protein Data Bank/BioMagResBank databases under the accession codes 2KMZ, 2KN0 and 2KN1 and 17237, 17247 and 17252. STRUCTURED DIGITAL ABSTRACT: TWEAK binds to hFn14 by surface plasmon resonance (View interaction) xeFn14 binds to TWEAK by enzyme linked immunosorbent assay (View interaction) TWEAK binds to xeFn14 by surface plasmon resonance (View interaction) hFn14 binds to TWEAK by enzyme linked immunosorbent assay (View interaction).
Subject(s)
Receptors, Tumor Necrosis Factor/chemistry , Tumor Necrosis Factors/chemistry , Xenopus Proteins/chemistry , Amino Acid Sequence , Animals , Cytokine TWEAK , Extracellular Space/chemistry , Humans , Models, Molecular , Molecular Sequence Data , Mutagenesis , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , TWEAK Receptor , Xenopus laevisABSTRACT
BIO8898 is one of several synthetic organic molecules that have recently been reported to inhibit receptor binding and function of the constitutively trimeric tumor necrosis factor (TNF) family cytokine CD40 ligand (CD40L, aka CD154). Small molecule inhibitors of protein-protein interfaces are relatively rare, and their discovery is often very challenging. Therefore, to understand how BIO8898 achieves this feat, we characterized its mechanism of action using biochemical assays and X-ray crystallography. BIO8898 inhibited soluble CD40L binding to CD40-Ig with a potency of IC(50) = 25 µM and inhibited CD40L-dependent apoptosis in a cellular assay. A co-crystal structure of BIO8898 with CD40L revealed that one inhibitor molecule binds per protein trimer. Surprisingly, the compound binds not at the surface of the protein but by intercalating deeply between two subunits of the homotrimeric cytokine, disrupting a constitutive protein-protein interface and breaking the protein's 3-fold symmetry. The compound forms several hydrogen bonds with the protein, within an otherwise hydrophobic binding pocket. In addition to the translational splitting of the trimer, binding of BIO8898 was accompanied by additional local and longer-range conformational perturbations of the protein, both in the core and in a surface loop. Binding of BIO8898 is reversible, and the resulting complex is stable and does not lead to detectable dissociation of the protein trimer. Our results suggest that a set of core aromatic residues that are conserved across a subset of TNF family cytokines might represent a generic hot-spot for the induced-fit binding of trimer-disrupting small molecules.
Subject(s)
CD40 Ligand/antagonists & inhibitors , Pyridines/pharmacology , Pyrrolidines/pharmacology , Animals , CD40 Antigens/immunology , CD40 Antigens/isolation & purification , CD40 Ligand/immunology , CD40 Ligand/isolation & purification , Cell Line , Cricetinae , Crystallography, X-Ray , Humans , Immunoglobulin Fc Fragments/immunology , Immunoglobulin Fc Fragments/isolation & purification , Models, Molecular , Molecular Structure , Molecular Weight , Protein Binding/drug effects , Pyridines/chemical synthesis , Pyridines/chemistry , Pyrrolidines/chemical synthesis , Pyrrolidines/chemistryABSTRACT
TWEAK, a TNF family ligand with pleiotropic cellular functions, was originally described as capable of inducing tumor cell death in vitro. TWEAK functions by binding its receptor, Fn14, which is up-regulated on many human solid tumors. Herein, we show that intratumoral administration of TWEAK, delivered either by an adenoviral vector or in an immunoglobulin Fc-fusion form, results in significant inhibition of tumor growth in a breast xenograft model. To exploit the TWEAK-Fn14 pathway as a therapeutic target in oncology, we developed an anti-Fn14 agonistic antibody, BIIB036. Studies described herein show that BIIB036 binds specifically to Fn14 but not other members of the TNF receptor family, induces Fn14 signaling, and promotes tumor cell apoptosis in vitro. In vivo, BIIB036 effectively inhibits growth of tumors in multiple xenograft models, including colon (WiDr), breast (MDA-MB-231), and gastric (NCI-N87) tumors, regardless of tumor cell growth inhibition response observed to BIIB036 in vitro. The anti-tumor activity in these cell lines is not TNF-dependent. Increasing the antigen-binding valency of BIB036 significantly enhances its anti-tumor effect, suggesting the contribution of higher order cross-linking of the Fn14 receptor. Full Fc effector function is required for maximal activity of BIIB036 in vivo, likely due to the cross-linking effect and/or ADCC mediated tumor killing activity. Taken together, the anti-tumor properties of BIIB036 validate Fn14 as a promising target in oncology and demonstrate its potential therapeutic utility in multiple solid tumor indications.
Subject(s)
Antineoplastic Agents , Apoptosis/drug effects , Neoplasms/therapy , Receptors, Tumor Necrosis Factor/agonists , Tumor Necrosis Factors/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/immunology , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cytokine TWEAK , HT29 Cells , Humans , Ligands , Mice , Neoplasms/immunology , Protein Binding , Receptors, Tumor Necrosis Factor/immunology , Receptors, Tumor Necrosis Factor/metabolism , TWEAK Receptor , Tumor Necrosis Factors/metabolism , Xenograft Model Antitumor AssaysABSTRACT
LTbetaR is a member of the TNF receptor family of proteins. It binds to two different cell surface ligands, LIGHT, a homotypic trimer, and LTalpha1beta2, a heterotypic trimer. We have measured the affinities of the dimeric IgG fusion protein, LTbetaRIgG, and monomeric LTbetaR protein binding to both LIGHT and LTalpha1beta2 using surface plasmon resonance and found values of <0.1 and 38 nM for LIGHT and <0.1 and 48 nM for LTalpha1beta2, respectively. We also determined the stoichiometries of binding for both forms of the receptor LTbetaRIgG and LTbetaR binding to LIGHT. The data obtained from several biophysical methods are consistent with receptor polypeptide to trimeric ligand ratios of 2:1. The determined masses of the complexes using SEC-LS corresponded to a single LTbetaRIgG bound to a LIGHT trimer, or two LTbetaR bound per LIGHT. Sedimentation velocity of varied ratios of LTbetaR to a fixed concentration of LIGHT were analyzed by SEDANAL and were successfully fit with a model with two tight binding sites on LIGHT and one poor affinity site. Isothermal calorimetric titration of LIGHT with either LTbetaR or LTbetaRIgG also demonstrated stoichiometries of 1:2 and 1:1, respectively. The binding of LTbetaR to LIGHT was endothermic and, hence, entropy-driven. TNFR p55 (extracellular domain) complexed with the trimeric ligand, TNFbeta, exhibits a 3:1 receptor/ligand stoichiometry. This complex has been used as the prototypical model setting the receptor-ligand complexation paradigm for the entire TNF family. The LTbetaR/LIGHT binding stoichiometry of 2:1 demonstrated here does not fit the paradigm. This has numerous implications for cell biology including signaling requiring only dimerization of LTbetaR rather than trimerization as expected from the structural paradigm.
Subject(s)
Lymphotoxin-alpha/chemistry , Lymphotoxin-alpha/metabolism , Receptors, Tumor Necrosis Factor/chemistry , Receptors, Tumor Necrosis Factor/metabolism , Binding Sites , Calorimetry/methods , Cell Physiological Phenomena , Chromatography, Gel , Dimerization , Electrophoresis, Polyacrylamide Gel , Immunoglobulin G/metabolism , Ligands , Models, Molecular , Molecular Weight , Recombinant Fusion Proteins/metabolism , Surface Plasmon Resonance , ThermodynamicsABSTRACT
The oligomeric state of BAFF (B cell activing factor), a tumor necrosis factor (TNF) family cytokine that plays a critical role in B cell development and survival, has been the subject of recent debate. Myc-tagged BAFF starting at residue Gln136 was previously reported to crystallize as trimers at pH 4.5, whereas a histidine-tagged construct of BAFF, starting at residue Ala134, formed a virus-like cluster containing 60 monomers when crystallized at pH 9.0. The formation of the BAFF 60-mer was pH dependent, requiring pH >or= 7.0. More recently, 60-mer formation was suggested to be artificially induced by the histidine tag, and it was proposed that BAFF, like all other TNF family members, is trimeric. We report here that a construct of BAFF with no amino-terminal tag (Ala134-BAFF) can form a 60-mer in solution. Using size exclusion chromatography and static light scattering to monitor trimer to 60-mer ratios in BAFF preparations, we find that 60-mer formation is pH-dependent and requires histidine 218 within the DE loop of BAFF. Biacore measurements established that the affinity of Ala134-BAFF for the BAFF receptor BAFFR/BR3 is similar to that of myc-Gln136-BAFF, which is exclusively trimeric in solution. However, Ala134-BAFF is more efficacious than myc-Gln136-BAFF in inducing B cell proliferation in vitro. We additionally show that BAFF that is processed and secreted by 293T cells transfected with full-length BAFF, or by a histiocytic lymphoma cell line (U937) that expresses BAFF endogenously, forms a pH-dependent 60-mer in solution. Our results indicate that the formation of the 60-mer in solution by the BAFF extracellular domain is an intrinsic property of the protein, and therefore that this more active form of BAFF may be physiologically relevant.
Subject(s)
Membrane Proteins/physiology , Protein Structure, Quaternary , Tumor Necrosis Factor-alpha/physiology , Animals , B-Cell Activating Factor , Chromatography, Gel , Humans , Hydrogen-Ion Concentration , Light , Mice , Molecular Weight , Pichia/metabolism , Scattering, RadiationABSTRACT
BAFF (B cell activating factor of the TNF family, also known as BlyS and TALL-1), a TNF family cytokine critical for the development and function of B cells, has been reported to bind to three receptors, BCMA (B cell maturation protein), TACI (transmembrane activator and CAML [calcium-modulator and cyclophilin ligand] interactor), and BAFFR (BAFF receptor), but with widely conflicting values for the affinity and selectivity of binding. BCMA and TACI additionally bind APRIL (a proliferation-inducing ligand), the TNF family ligand most homologous to BAFF. Using soluble, monomeric forms of the receptors, we demonstrate that BAFFR binds BAFF with K(D) approximately 16 nM, while BCMA binds with K(D) approximately 1.6 microM, indicating a approximately 100-fold selectivity for binding to BAFFR over BCMA. APRIL shows the opposite selectivity, binding to BCMA with K(D) approximately 16 nM while showing no detectable affinity for BAFFR (K(D) > 3 microM). The binding of BAFF or APRIL to these receptors is highly sensitive to assay-dependent avidity effects, likely explaining the widely ranging affinity values reported in the literature. Binding of BAFF to BCMA-Fc, a bivalent fusion protein consisting of the extracellular domain of BCMA fused to the hinge and CH1 and CH2 domains of human IgG1, in solution or coated onto an ELISA plate gave apparent binding affinities of approximately 0.63 and approximately 0.15 nM, respectively, compared to values of K(D(app)) Subject(s)
B-Lymphocytes/metabolism
, Membrane Proteins/metabolism
, Receptors, Tumor Necrosis Factor/metabolism
, Tumor Necrosis Factor-alpha/metabolism
, Animals
, B-Cell Activating Factor
, B-Cell Activation Factor Receptor
, B-Cell Maturation Antigen
, Cell Line
, Cell Proliferation
, Enzyme-Linked Immunosorbent Assay
, Humans
, Ligands
, Mice
, Mice, Inbred C57BL
, Protein Binding
, Solutions
, Spleen/cytology
, Surface Plasmon Resonance
, Tumor Necrosis Factor Ligand Superfamily Member 13
ABSTRACT
To clarify whether glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFRalpha1), the glycosylphosphatidylinositol (GPI)-linked coreceptor for GDNF, is also a functional coreceptor for artemin (ART), we have studied receptor binding, signaling, and neuronal survival. In cell-free binding studies, GFRalpha1-Ig displayed strong preferential binding to GDNF, though in the presence of soluble RET, weak binding to ART could also be detected. However, using GFRalpha1-transfected NB41A3 cells, ART showed no detectable competition against the binding of (125)I-labeled GDNF. Moreover, ART failed to induce phosphorylation of extracellular signal-related kinase (ERK) and Akt in these cells and was >10(4)-fold less potent than GDNF in stimulating RET phosphorylation. When rat primary dorsal root ganglion (DRG) neurons were used, only the survival promoting activity of GDNF and not that of ART was blocked by an anti-GFRalpha1 antibody. These results indicate that although ART can interact weakly with soluble GFRalpha1 constructs under certain circumstances in vitro, in cell-based functional assays GFRalpha1 is at least 10 000-fold selective for GDNF over ART. The extremely high selectivity of GFRalpha1 for GDNF over ART and the low reactivity of ART for this receptor suggest that GFRalpha1 is not likely to be a functional coreceptor for ART in vivo.
Subject(s)
Nerve Growth Factors/metabolism , Nerve Tissue Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Animals , Cell Line , Cell Line, Tumor , Cell Membrane/metabolism , Cell Survival/physiology , Ganglia, Spinal/cytology , Ganglia, Spinal/physiology , Glial Cell Line-Derived Neurotrophic Factor , Glial Cell Line-Derived Neurotrophic Factor Receptors , Humans , Mice , Nerve Growth Factors/physiology , Nerve Tissue Proteins/physiology , Neurons, Afferent/cytology , Neurons, Afferent/physiology , Phosphorylation , Protein Binding , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-ret , Rats , Rats, Sprague-Dawley , Receptor Protein-Tyrosine Kinases/physiology , Signal Transduction/genetics , Solutions , Surface Plasmon Resonance , TransfectionABSTRACT
We have identified a small-molecule inhibitor of tumor necrosis factor alpha (TNF-alpha) that promotes subunit disassembly of this trimeric cytokine family member. The compound inhibits TNF-alpha activity in biochemical and cell-based assays with median inhibitory concentrations of 22 and 4.6 micromolar, respectively. Formation of an intermediate complex between the compound and the intact trimer results in a 600-fold accelerated subunit dissociation rate that leads to trimer dissociation. A structure solved by x-ray crystallography reveals that a single compound molecule displaces a subunit of the trimer to form a complex with a dimer of TNF-alpha subunits.
Subject(s)
Indoles/chemistry , Indoles/pharmacology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/chemistry , Biotinylation , Chemical Phenomena , Chemistry, Physical , Crystallography, X-Ray , Dimerization , Fluorescence , Hydrogen/chemistry , Hydrophobic and Hydrophilic Interactions , Indoles/chemical synthesis , Kinetics , Mass Spectrometry , Models, Chemical , Models, Molecular , Molecular Conformation , Molecular Structure , Protein Conformation , Protein Subunits/chemistry , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
To gauge the experimental variability associated with Biacore analysis, 36 different investigators analyzed a small molecule/enzyme interaction under similar conditions. Acetazolamide (222 g/mol) binding to carbonic anhydrase II (CAII; 30000 Da) was chosen as a model system. Both reagents were stable and their interaction posed a challenge to measure because of the low molecular weight of the analyte and the fast association rate constant. Each investigator created three different density surfaces of CAII and analyzed an identical dilution series of acetazolamide (ranging from 4.1 to 1000 nM). The greatest variability in the results was observed during the enzyme immobilization step since each investigator provided their own surface activating reagents. Variability in the quality of the acetazolamide binding responses was likely a product of how well the investigators' instruments had been maintained. To determine the reaction kinetics, the responses from the different density surfaces were fit globally to a 1:1 interaction model that included a term for mass transport. The averaged association and dissociation rate constants were 3.1+/-1.6 x 10(6)M(-1)s(-1) and 6.7+/-2.5 x 10(-2)s(-1), respectively, which corresponded to an average equilibrium dissociation constant (K(D) of 2.6+/-1.4 x 10(-8)M. The results provide a benchmark of variability in interpreting binding constants from the biosensor and highlight keys areas that should be considered when analyzing small molecule interactions.