Ligand-induced assembling of the type I interferon receptor on supported lipid bilayers

Type I interferons (IFNs) elicit antiviral, antiproliferative and immuno-modulatory responses through binding to a shared receptor consisting of the transmembrane proteins ifnar1 and ifnar2. Differential signaling by different interferons, in particular IFNalphas and IFNbeta, suggests different modes of receptor engagement. Using reflectometric interference spectroscopy (RIfS), we studied kinetics and affinities of the interactions between IFNs and the extracellular receptor domains of ifnar1 (ifnar1-EC) and ifnar2 (ifnar2-EC). For IFNalpha2, we determined a K(D) value of 3 nM and 5 microM for the interaction with ifnar2-EC and ifnar1-EC, respectively. As compared to IFNalpha2, IFNbeta formed complexes with ifnar2-EC as well as ifnar1-EC with substantially higher affinity. For neither IFNalpha2 nor IFNbeta was stabilization of the complex with ifnar1-EC in the presence of soluble ifnar2-EC observed. We investigated ligand-induced complex formation with ifnar1-EC and ifnar2-EC being tethered onto solid-supported, fluid lipid bilayers by RIfS and total internal reflection fluorescence spectroscopy. We observed very stable binding of IFNalpha2 at high receptor surface concentrations with an apparent k(d) value approximately 200 times lower than that for ifnar2-EC alone. The apparent k(d) value was strongly dependent on the surface concentration of the receptor components, suggesting kinetic stabilization. This was corroborated by the fast exchange of labeled IFNalpha2 bound to the receptor by unlabeled IFNalpha2. Taken together, our results indicate that IFN first binds to ifnar2 and subsequently recruits ifnar1 in a transient fashion. In particular, this second step is much more efficient for IFNbeta than for IFNalpha2, which could explain differential activities observed for these IFNs.     

Differential receptor subunit affinities of type I interferons govern differential signal activation

Type I interferons (IFNs) elicit antiviral, antiproliferative and immunmodulatory responses by binding to a shared cell surface receptor comprising the transmembrane proteins ifnar1 and ifnar2. Activation of differential response patterns by IFNs has been observed, suggesting that members of the family play different roles in innate immunity. The molecular basis for differential signaling has not been identified yet. Here, we have investigated the recognition of various IFNs including several human IFNalpha species, human IFNomega and human IFNbeta as well as ovine IFNtau2 by the receptor subunits in detail. Binding to the extracellular domains of ifnar1 (ifnar1-EC) and ifnar2 (ifnar2-EC) was monitored in real time by reflectance interference and total internal reflection fluorescence spectroscopy. For all IFNs investigated, competitive 1:1 interaction not only with ifnar2-EC but also with ifnar1-EC was shown. Furthermore, ternary complex formation was studied with ifnar1-EC and ifnar2-EC tethered onto solid-supported membranes. These analyses confirmed that the signaling complexes recruited by IFNs have very similar architectures. However, differences in rate and affinity constants over several orders of magnitude were observed for both the interactions with ifnar1-EC and ifnar2-EC. These data were correlated with the potencies of ISGF3 activation, antiviral and anti-proliferative activity on 2fTGH cells. The ISGF3 formation and antiviral activity correlated very well with the binding affinity towards ifnar2. In contrast, the affinity towards ifnar1 played a key role for antiproliferative activity. A striking correlation was observed for relative binding affinities towards ifnar1 and ifnar2 with the differential antiproliferative potency. This correlation was confirmed by systematically engineering IFNalpha2 mutants with very high differential antiproliferative potency.     

Fast transient cytokine-receptor interactions monitored in real time by reflectometric interference spectroscopy

Investigating protein-protein interactions by mutational analysis requires practical techniques for quantifying rate constants and equilibrium constants over several orders of magnitude with reasonably high sample throughput. We have employed spectroscopic interferometry for label-free monitoring of the interaction between the cytokine interferon alpha2 (IFNalpha2) and the extracellular domain of its receptor ifnar2 (ifnar2-EC). We implemented a versatile surface chemistry for the glass substrate of this transducer for covalent immobilization of proteins. Affinity capturing with a monoclonal anti-ifnar2-EC antibody (mAb) followed by crosslinking with a second, noncompetitive mAb provided stable, but still reversible, immobilization of ifnar2-EC. We measured kinetics and affinity of numerous of mutants of IFNalpha2 and ifnar2-EC. Dissociation rate constants up to 0.3 s(-1) and association rate constants up to 3 x 10(6) M(-)1 s(-1) were resolved by the system. Dissociation constants down to 200 microM were measured with protein concentrations up to 50 microM without no background signal or nonspecific binding. The instrument detection limit is approximately 10 pm without the need for temperature stabilization or referencing channels. The system proved effective for large-scale mutational analysis involving alanine scanning mutagenesis and double mutant cycles.     

Lateral ligand-receptor interactions on membranes probed by simultaneous fluorescence-interference detection

We describe an experimental approach for studying ligand-receptor interactions in the plane of the membrane. The extracellular domains of the type I interferon receptor subunits ifnar1-EC and ifnar2-EC were tethered in an oriented fashion onto solid-supported, fluid lipid bilayers, thus mimicking membrane anchoring and lateral diffusion of the receptor. Ligand-induced receptor assembling was investigated by simultaneous total internal reflection fluorescence spectroscopy and reflectance interferometry (RIf). Based on a rigorous characterization of the interactions of fluorescence-labeled IFNalpha2 with each of the receptor subunits, the dynamics of the ternary complex formation on the fluid lipid bilayer was addressed in further detail making use of the features of the simultaneous detection. All these measurements supported the formation of a ternary complex in two steps, i.e., association of the ligand to ifnar2-EC and subsequent recruitment of ifnar1-EC on the surface of the membrane. Based on the ability to control and quantify the receptor surface concentrations, equilibrium, and rate constants of the interaction in the plane of the membrane were determined by monitoring ligand dissociation at different receptor surface concentrations. Using mutants of IFNalpha2 binding to ifnar2-EC with different association rate constants, the key role of the association rate constants for the assembling mechanism was demonstrated.     

Mutational and structural analysis of the binding interface between type I interferons and their receptor Ifnar2.

Type I interferons (IFN) exert pleiotropic activities through binding to two cell surface receptors, ifnar1 and ifnar2. We are investigating the biophysical basis of IFN signaling by characterizing the complex of the extra-cellular domain of ifnar2 (ifnar2-EC) with IFNs on the level of purified recombinant proteins in vitro. Here, we present a detailed mutational study on the functional epitopes on both IFN and ifnar2. Kinetic and thermodynamic parameters were determined by label-free heterogeneous phase detection. On IFNalpha2, a relatively small functional epitope comprising ten amino acid residues was localized, which is nearly entirely formed by residues on the AB loop. Two hot-spot residues, L30 and R33, account for two-thirds of the total interaction energy. Comparing the anti-viral potency of the various mutants to the binding affinity towards ifnar2 revealed a proportional correlation between the two, suggesting a rate-limiting role of ifnar2 binding in IFN signaling. On ifnar2, residues T46, I47 and M48 were identified as hot-spots in the interaction with IFNalpha2. For another ten residues on ifnar2, significant contribution of interaction energy was determined. Based on these data, the functional epitope on ifnar2 was defined according to a homology model based on other members of the class II hCR family in good agreement with the complementary functional epitope on IFNalpha2. Although IFNalpha2 and IFNbeta bind competitively to the same functional epitope, mutational analysis revealed distinct centers of binding for these IFNs on ifnar2. This small shift of the binding site may result in different angular orientation, which can be critically coupled to cytoplasmic signaling.     

Determination of the two-dimensional interaction rate constants of a cytokine receptor complex

Ligand-receptor interactions within the plane of the plasma membrane play a pivotal role for transmembrane signaling. The biophysical principles of protein-protein interactions on lipid bilayers, though, have hardly been experimentally addressed. We have dissected the interactions involved in ternary complex formation by ligand-induced cross-linking of the subunits of the type I interferon (IFN) receptors ifnar1 and ifnar2 in vitro. The extracellular domains ifnar1-ectodomain (EC) and ifnar2-EC were tethered in an oriented manner on solid-supported lipid bilayers. The interactions of IFNalpha2 and several mutants, which exhibit different association and dissociation rate constants toward ifnar1-EC and ifnar2-EC, were monitored by simultaneous label-free detection and surface-sensitive fluorescence spectroscopy. Surface dissociation rate constants were determined by measuring ligand exchange kinetics, and by measuring receptor exchange on the surface by fluorescence resonance energy transfer. Strikingly, approximately three-times lower dissociation rate constants were observed for both receptor subunits compared to the dissociation in solution. Based on these directly determined surface-dissociation rate constants, the surface-association rate constants were assessed by probing ligand dissociation at different relative surface concentrations of the receptor subunits. In contrast to the interaction in solution, the association rate constants depended on the orientation of the receptor components. Furthermore, the large differences in association kinetics observed in solution were not detectable on the surface. Based on these results, the key roles of orientation and lateral diffusion on the kinetics of protein interactions in plane of the membrane are discussed.     

Optimizing the binding affinity of a carrier protein: a case study on the interaction between soluble ifnar2 and interferon beta

Prolonging the circulatory half-life of low mass protein drugs can be achieved either by administration of a pro-drug or through co-injection with a carrier protein that will slowly release the active protein. The rate of release is concentration and affinity dependent. The optimal relationship between these two in prolonging the half-life of a pro-drug is the focus of this work. Interferon (IFN) beta is one of the most widely used protein drugs in the clinic. Here, we show that the circulatory half-life of IFNbeta can be significantly extended by co-administration with the extracellular domain of the IFN receptor ifnar2 (ifnar2-EC). To investigate the concentration/affinity relation, a range of tighter binding ifnar2-EC mutants was designed that bind IFNbeta, but not IFNalpha2, up to 50-fold tighter compared with the wild-type ifnar2-EC. This increased affinity is related to a slower dissociation rate, whereas the association of IFNbeta with ifnar2-EC is already near optimum. Using the wild-type and mutant receptors, we investigated their potential in occluding IFNbeta from circulation in a tissue culture assay, as well as in rats. To determine the potential of ifnar2-EC as a carrier protein, we co-administered a mixture of IFNbeta and ifnar2-EC to rats both intravenously and subcutaneously, and followed the blood plasma concentrations of IFNbeta over time. The tighter binding ifnar2-EC mutant had a clear advantage in prolonging the half-life of IFNbeta in circulation, even when lower protein concentrations were administered. A numerical simulation of the in vivo data demonstrates that the optimal binding affinity of a carrier protein is around the concentration needed to obtain optimal activity of the ligand.     

Ligand binding induces a conformational change in ifnar1 that is propagated to its membrane-proximal domain

The type I interferon (IFN) receptor plays a key role in innate immunity against viral and bacterial infections. Here, we show by intramolecular Förster resonance energy transfer spectroscopy that ligand binding induces substantial conformational changes in the ectodomain of ifnar1 (ifnar1-EC). Binding of IFNα2 and IFNβ induce very similar conformations of ifnar1, which were confirmed by single-particle electron microscopy analysis of the ternary complexes formed by IFNα2 or IFNβ with the two receptor subunits ifnar1-EC and ifnar2-EC. Photo-induced electron-transfer-based fluorescence quenching and single-molecule fluorescence lifetime measurements revealed that the ligand-induced conformational change in the membrane-distal domains of ifnar1-EC is propagated to its membrane-proximal domain, which is not involved in ligand recognition but is essential for signal activation. Temperature-dependent ligand binding studies as well as stopped-flow fluorescence experiments corroborated a multistep conformational change in ifnar1 upon ligand binding. Our results thus suggest that the relatively intricate architecture of the type I IFN receptor complex is designed to propagate the ligand binding event to and possibly even across the membrane by conformational changes.     

Functional cartography of the ectodomain of the type I interferon receptor subunit ifnar1

Ligand-induced cross-linking of the type I interferon (IFN) receptor subunits ifnar1 and ifnar2 induces a pleiotrophic cellular response. Several studies have suggested differential signal activation by flexible recruitment of the accessory receptor subunit ifnar1. We have characterized the roles of the four Ig-like sub-domains (SDs) of the extracellular domain of ifnar1 (ifnar1-EC) for ligand recognition and receptor assembling. Various sub-fragments of ifnar1-EC were expressed in insect cells and purified to homogeneity. Solid phase binding assays with the ligands IFN(alpha)2 and IFN(beta) revealed that all three N-terminal SDs were required and sufficient for ligand binding, and that IFN(alpha)2 and IFN(beta) compete for this binding site. Cellular binding assays with different fragments, however, highlighted the key role of the membrane-proximal SD for the formation of an in situ IFN-receptor complex. Even substitution with the corresponding SD from homologous cytokine receptors did not restore high-affinity ligand binding. Receptor assembling analysis on supported lipid bilayers in vitro revealed that the membrane-proximal SD controls appropriate orientation of the receptor on the membrane, which is required for efficient association of ifnar1 into the ternary complex.     

Systematic mutational mapping of sites on human interferon-beta-1a that are important for receptor binding and functional activity

A systematic mutational analysis of human interferon-beta-1a (IFN-beta) was performed to identify regions on the surface of the molecule that are important for receptor binding and for functional activity. The crystal structure of IFN-beta-1a was used to design a panel of 15 mutant proteins, in each of which a contiguous group of 2-8 surface residues was mutated, in most instances to alanine. The mutants were analyzed for activity in vitro in antiviral and in antiproliferation assays, and for their ability to bind to the type I IFN (ifnar1/ifnar2) receptor on Daudi cells and to a soluble ifnar2 fusion protein (ifnar2-Fc). Abolition of binding to ifnar2-Fc for mutants A2, AB1, AB2, and E established that the ifnar2 binding site on IFN-beta comprises parts of the A helix, the AB loop, and the E helix. Mutations in these areas, which together define a contiguous patch of the IFN-beta surface, also resulted in reduced affinity for binding to the receptor on cells and in reductions in activity of 5-50-fold in functional assays. A second receptor interaction site, concluded to be the ifnar1 binding site, was identified on the opposite face of the molecule. Mutations in this region, which encompasses parts of the B, C, and D helices and the DE loop, resulted in disparate effects on receptor binding and on functional activity. Analysis of antiproliferation activity as a function of the level of receptor occupancy allowed mutational effects on receptor activation to be distinguished from effects on receptor binding. The results suggest that the binding energy from interaction of IFN-beta with ifnar2 serves mainly to stabilize the bound IFN/receptor complex, whereas the binding energy generated by interaction of certain regions of IFN-beta with ifnar1 is not fully expressed in the observed affinity of binding but instead serves to selectively stabilize activated states of the receptor.     

Determination of the human type I interferon receptor binding site on human interferon-alpha2 by cross saturation and an NMR-based model of the complex

Type I interferons (IFNs) are a family of homologous helical cytokines that exhibit pleiotropic effects on a wide variety of cell types, including antiviral activity and antibacterial, antiprozoal, immunomodulatory, and cell growth regulatory functions. Consequently, IFNs are the human proteins most widely used in the treatment of several kinds of cancer, hepatitis C, and multiple sclerosis. All type I IFNs bind to a cell surface receptor consisting of two subunits, IFNAR1 and IFNAR2, associating upon binding of interferon. The structure of the extracellular domain of IFNAR2 (R2-EC) was solved recently. Here we study the complex and the binding interface of IFNalpha2 with R2-EC using multidimensional NMR techniques. NMR shows that IFNalpha2 does not undergo significant structural changes upon binding to its receptor, suggesting a lock-and-key mechanism for binding. Cross saturation experiments were used to determine the receptor binding site upon IFNalpha2. The NMR data and previously published mutagenesis data were used to derive a docking model of the complex with an RMSD of 1 Angstrom, and its well-defined orientation between IFNalpha2 and R2-EC and the structural quality greatly improve upon previously suggested models. The relative ligand-receptor orientation is believed to be important for interferon signaling and possibly one of the parameters that distinguish the different IFN I subtypes. This structural information provides important insight into interferon signaling processes and may allow improvement in the development of therapeutically used IFNs and IFN-like molecules.     

NMR backbone dynamics of the human type I interferon binding subunit, a representative cytokine receptor

The antiviral and antiproliferative activities of type I interferons (IFNs) are mediated by a common receptor, and its second subunit (IFNAR2) exhibits nanomolar affinity to both IFNalpha and IFNbeta subtypes. We have previously determined the structure of the IFN-binding extracellular domain of IFNAR2 (IFNAR2-EC) using multidimensional NMR [Chill, J. H., Quadt, S. R., Levy, R., Schreiber, G. E., and Anglister, J. (2003) Structure 11, 791-802], showing it to comprise two fibronectin domains linked by a hinge. As the first cytokine receptor structure determined in the unliganded state and in solution, IFNAR2-EC offers an opportunity to characterize the dynamics of the cytokine receptor family and their correlation to biological function. Backbone dynamics of IFNAR2-EC were investigated using 15N relaxation at 11.74 and 18.79 T, and measurements of residual dipolar couplings (RDCs). Dynamics of the binding site distinguish between rigid structural domains, which stabilize the binding site conformation, and a more flexible binding interface which interacts with the ligand. Measurements of diffusional anisotropy and RDCs and model-free analysis all show that the backbone of the hinge interdomain region of IFNAR2-EC is rigid on the picosecond to nanosecond time scale. Signal transduction in cytokines receptors is initiated by ligand-induced juxtaposition of the two receptor subunits, triggering the mutual phosphorylation of kinases associated to their cytoplasmic domains. The rigidity of the hinge ensures correct positioning of the receptor subunits in the ternary signaling complex and modulates the interaction between kinases in the cytoplasm, thereby controlling the rate and efficiency of phosphorylation.     

Photodegradation mechanism and reaction kinetics of recombinant human interferon-alpha2a.

The photodegradation mechanism of recombinant human interferon-alpha2a (IFNalpha2a) has been investigated using absorption, fluorescence, and circular dichroism (CD) spectroscopies, and fluorescence photobleaching kinetics measurements under various conditions. After photobleaching, the absorption profile of aromatic amino acid residues in IFNalpha2a was almost absent, and an absorption profile showing a monotonic increase toward short wavelengths was observed. According to the CD spectrum analysis, partial unfolding of IFNalpha2a was accompanied by a complete loss of fluorescence. This unfolding was attributed to tryptophan-mediated photoinduced disulfide bond cleavage. Photooxygenation and photoionization of tryptophan (Trp) residues followed by subsequent radical reactions were the main photodegradation pathways of IFNalpha2a. Photobleaching kinetics was faster in acidic solution (pH 2.5) than in neutral solution (pH 7.4). The variation of photobleaching kinetics seemed to be caused by the structural differences in IFNalpha2a according to the solution pH. The relationship between the protein conformation and photobleaching rate could be explained based on the competition between excited state energy transfer and the photoionization process in Trp residues.     

Protein dynamics: imidazole and 2-mercaptoethanol binding to the Rhodobacter capsulatus cytochrome c(2) mutant,glycine 95 proline

The Class I c-type cytochromes can bind exogenous ligands in the oxidized state, with the kinetics of ligand binding providing information on naturally occurring intramolecular dynamics. Typically, nitrogenous bases are used as ligands; however, it is less well known that 2-mercaptoethanol (BME), a commonly used cytochrome reducing agent, can form a complex with the heme. To better understand the cytochrome-mercaptan interaction, we have investigated the kinetics of binding of BME to wild type and mutants of Rhodobacter capsulatus cytochrome c(2) and to horse cytochrome c. Complex formation with the G95P mutant is apparent from the formation of a green color and a shift in the Soret peak to 418 nm from 410 nm upon addition of BME. Unlike horse cytochrome c and wild-type R. capsulatus cytochrome c(2), G95P permits the kinetics of formation of the BME-G95P complex to be measured since complex formation and reduction kinetics can be resolved. The affinity constant for the binding of BME to mutant G95P was strong ( approximately 1.5 x 10(5)M(-1)) and the kinetics of formation of the BME-G95P complex were found to undergo a change in rate-limiting step consistent with a concentration-independent protein rearrangement (68s(-1)) followed by second-order binding of BME ( approximately approximately 1.3 x 10(5)M(-1)s(-1)). The most remarkable characteristic of mutant G95P is the relatively large amount of high-spin species in equilibrium with the low- spin form, which can be estimated to be approximately 3% at pH 7. The BME binding kinetics, coupled with the kinetics of imidazole binding to G95P, allow us, for the first time, to specify all four rate constants describing the ligand binding reaction. Moreover, we can use the kinetic results to estimate the rate constants for ligand binding with the wild-type cytochrome c(2). This has also allowed us to quantify and more fully interpret cytochrome dynamics.     

The human interferon receptor: NMR-based modeling, mapping of the IFN-alpha 2 binding site, and observed ligand-induced tightening

The human interferon receptor (IFNAR) mediates the antiviral and antiproliferative activities of type I interferons (IFNs). This receptor is comprised of subunits IFNAR1 and IFNAR2, the latter exhibiting nanomolar affinity for IFNs. Here the extracellular domain of IFNAR2 (IFNAR2-EC), a soluble 25 kDa IFN-binding polypeptide, and its complex with IFN-alpha 2 were studied using multidimensional NMR. IFNAR2-EC is comprised of two fibronectin-III (FN-III) domains connected by a helical hinge region. The deduced global fold was utilized to improve the alignment of IFNAR2-EC against structurally related receptors and to model its structure. A striking feature of IFNAR2-EC is the limited and localized deviations in chemical shifts exhibited upon ligand binding, observed for only 15% of its backbone (1)H and (15)N nuclei. Analysis of these deviations maps the IFN-alpha 2 binding site upon IFNAR2-EC to a contiguous surface on the N-terminal domain, including the S3-S4 loop (residues 44-53), the S5-S6 loop and S6 beta-strand (residues 74-82), and the S7 beta-strand and the hinge region (residues 95-105). The C-terminal domain contributes only marginally to ligand binding, and no change in the hypothesized interdomain interface is observed. The proposed binding domain encompasses all residues implicated by mutagenesis studies in IFN binding, and suggests adjacent residues cooperate in forming the binding surface. D(2)O-exchange experiments indicate that binding of IFN-alpha2 induces tightening of the N-terminal domain of IFNAR2-EC. This increase in receptor rigidity may play an important role in initiating the intracellular stage of the IFN signaling cascade.     

Structural, kinetic, and thermodynamic analysis of the binding of the 40 kDa PEG-interferon-alpha2a and its individual positional isomers to the extracellular domain of the receptor IFNAR2

Type-I Interferons exert antiviral and antiproliferative activities through the binding to a common cell surface receptor comprising two subunits, IFNAR1 and IFNAR2. Human recombinant Interferon-alpha(2a) (IFNalpha(2a)) is a potent drug (Roferon-A) used to treat various cancers and viral diseases including Hepatitis B/C infections. To significantly improve the pharmacological properties of the drug, a pegylated form of IFNalpha(2a) was developed (PEGASYS). This 40 kDa PEG-conjugated IFNalpha(2a) ((40)PEG-IFNalpha(2a)) is obtained by the covalent binding of one 40 kDa branched PEG-polymer to a lysine side-chain of IFNalpha(2a). Here, we report the detailed structural, kinetic, and thermodynamic analysis of the binding to the extracellular domain of the receptor IFNAR2 of (40)PEG-IFNalpha(2a) and its isolated positional isomers modified at K31, K134, K131, K121, K164, and K70, respectively, in comparison with unmodified IFNalpha(2a). Our binding studies, using the surface plasmon resonance technique, show that the pegylation does not abolish the binding to the receptor, but significantly reduces the affinity mainly due to a change of the association rate. The results are supported by modeling and simulation of the binding, using Self-Avoiding-Walk calculations for the polymer conformations. A correlation between the structural parameters and the kinetic and thermodynamic parameters of the binding of the positional isomers could be established. For the Isomer-K31 and -K164, the PEG-polymer attachment point is located in proximity to the binding interface, and the isomers display affinity in the range 150-520 nM in an enthalpy-driven binding process. In contrast for the Isomer-K134, -K131, -K121, and -K70, the PEG-polymer is attached remotely from the binding interface, and the isomers exhibit a higher affinity (32-76 nM) in an entropy-driven binding process. This study constitutes an essential collection of knowledge on which the interaction of (40)PEG-IFNalpha(2a) and its positional isomers with its cellular receptors can be better understood.