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• W2053641099 abstract "IL-7 and IL-7Rα bind the γc receptor, forming a complex crucial to several signaling cascades leading to the development and homeostasis of T and B cells. We report that the IL-7Rα ectodomain uses glycosylation to modulate its binding constants to IL-7, unlike the other receptors in the γc family. IL-7 binds glycosylated IL-7Rα 300-fold more tightly than unglycosylated IL-7Rα, and the enhanced affinity is attributed primarily to an accelerated on rate. Structural comparison of IL-7 in complex to both forms of IL-7Rα reveals that glycosylation does not participate directly in the binding interface. The SCID mutations of IL-7Rα locate outside the binding interface with IL-7, suggesting that the expressed mutations cause protein folding defects in IL-7Rα. The IL-7/IL-7Rα structures provide a window into the molecular recognition events of the IL-7 signaling cascade and provide sites to target for designing new therapeutics to treat IL-7-related diseases. IL-7 and IL-7Rα bind the γc receptor, forming a complex crucial to several signaling cascades leading to the development and homeostasis of T and B cells. We report that the IL-7Rα ectodomain uses glycosylation to modulate its binding constants to IL-7, unlike the other receptors in the γc family. IL-7 binds glycosylated IL-7Rα 300-fold more tightly than unglycosylated IL-7Rα, and the enhanced affinity is attributed primarily to an accelerated on rate. Structural comparison of IL-7 in complex to both forms of IL-7Rα reveals that glycosylation does not participate directly in the binding interface. The SCID mutations of IL-7Rα locate outside the binding interface with IL-7, suggesting that the expressed mutations cause protein folding defects in IL-7Rα. The IL-7/IL-7Rα structures provide a window into the molecular recognition events of the IL-7 signaling cascade and provide sites to target for designing new therapeutics to treat IL-7-related diseases. IL-7, IL-7Rα, and γc form a ternary complex that plays fundamental roles in extracellular matrix remodeling, development, and homeostasis of T and B cells (reviewed in Mazzucchelli and Durum, 2007Mazzucchelli R. Durum S.K. Interleukin-7 receptor expression: intelligent design.Nat. Rev. Immunol. 2007; 7: 144-154Crossref PubMed Scopus (438) Google Scholar). IL-7Rα also crossreacts to form a ternary complex with thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR), and activates the TSLP pathway, resulting in T and dentritic cell proliferation in humans and further B cell development in mice (Leonard, 2002Leonard W.J. TSLP: finally in the limelight.Nat. Immunol. 2002; 3: 605-607Crossref PubMed Scopus (99) Google Scholar). Tight regulation of the signaling cascades activated by the complexes is therefore crucial to normal cellular function. Understimulation of the IL-7 pathway caused by mutations in the IL-7Rα ectodomain inhibits T and B cell development, resulting in patients with a form of severe combined immunodeficiency (SCID) (Giliani et al., 2005Giliani S. Mori L. de Saint Basile G. Le Deist F. Rodriguez-Perez C. Forino C. Mazzolari E. Dupuis S. Elhasid R. Kessel A. et al.Interleukin-7 receptor α (IL-7Rα) deficiency: cellular and molecular bases. Analysis of clinical, immunological, and molecular features in 16 novel patients.Immunol. Rev. 2005; 203: 110-126Crossref PubMed Scopus (133) Google Scholar, Puel et al., 1998Puel A. Ziegler S.F. Buckley R.H. Leonard W.J. Defective IL7R expression in T(−)B(+)NK(+) severe combined immunodeficiency.Nat. Genet. 1998; 20: 394-397Crossref PubMed Scopus (638) Google Scholar). Overstimulation of the pathways leads to allergic rhinitis, autoimmunity, heart disease, and proliferation of cancers (reviewed in Sportes and Gress, 2007Sportes C. Gress R.E. Interleukin-7 immunotherapy.Adv. Exp. Med. Biol. 2007; 601: 321-333Crossref PubMed Scopus (23) Google Scholar). In clinical trials of patients with hepatitis and recovering cancer patients, IL-7 is being tested to spark T cell development and expansion (reviewed in Sportes and Gress, 2007Sportes C. Gress R.E. Interleukin-7 immunotherapy.Adv. Exp. Med. Biol. 2007; 601: 321-333Crossref PubMed Scopus (23) Google Scholar). IL-7 and IL-7Rα belong to the γc family of cytokine receptors, which includes IL-2, -4, -9, -15, -21, and receptors. The ILs and ectodomains of the receptors share <15% sequence identity with each other including the binding interfaces (see Figure S1 available online). Besides binding γc, all of the ILs and receptors in the γc family are glycoproteins. Despite the universal glycosylation of the γc family, glycosylation has not been vital to the binding interactions among family members. Studies show that glycosylation plays no role in complex formation between IL-2 (Rickert et al., 2004Rickert M. Boulanger M.J. Goriatcheva N. Garcia K.C. Compensatory energetic mechanisms mediating the assembly of signaling complexes between interleukin-2 and its α, β, and γ (c) receptors.J. Mol. Biol. 2004; 339: 1115-1128Crossref PubMed Scopus (63) Google Scholar), -4 (Hage et al., 1999Hage T. Sebald W. Reinemer P. Crystal structure of the interleukin-4/receptor α chain complex reveals a mosaic binding interface.Cell. 1999; 97: 271-281Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar), -15 (Matsumoto et al., 2003Matsumoto M. Misawa S. Tsumoto K. Kumagai I. Hayashi H. Kobayashi Y. On-column refolding and characterization of soluble human interleukin-15 receptor α-chain produced in Escherichia coli.Protein Expr. Purif. 2003; 31: 64-71Crossref PubMed Scopus (21) Google Scholar), -21 (Zhang et al., 2003Zhang J.L. Foster D. Sebald W. Human IL-21 and IL-4 bind to partially overlapping epitopes of common γ-chain.Biochem. Biophys. Res. Commun. 2003; 300: 291-296Crossref PubMed Scopus (29) Google Scholar), and receptors. Three Asns of IL-7 and six Asns of the IL-7Rα ectodomain may be N-linked glycosylated from the Asn-X-Ser/Thr recognition motif. Glycosylation of IL-7 does not affect its binding/function to IL-7Rα (Goodwin et al., 1989Goodwin R.G. Lupton S. Schmierer A. Hjerrild K.J. Jerzy R. Clevenger W. Gillis S. Cosman D. Namen A.E. Human interleukin 7: molecular cloning and growth factor activity on human and murine B-lineage cells.Proc. Natl. Acad. Sci. USA. 1989; 86: 302-306Crossref PubMed Scopus (245) Google Scholar). It remains an open question to the importance of glycosylation of the IL-7Rα in binding and function. To our knowledge for the first time among γc family members, we report that glycosylation is important to the interaction between IL-7 and IL-7Rα. We compare the binding constants of IL-7 to unglycosylated and glycosylated IL-7Rα and show that the enhanced binding affinity of IL-7 to glycosylated IL-7Rα results primarily from an accelerated on rate. Furthermore, we have determined the crystal structures of IL-7 complexes bound to unglycosylated IL-7Rα at 2.7 Å and glycosylated IL-7Rα at 2.9 Å. Glycosylation of IL-7Rα does not induce large conformational changes in the complexes and the glycans are located outside the IL-7/IL-7Rα binding interface, indicating an indirect mechanism of binding enhancement. The IL-7/IL-7Rα binding interface displays the smallest, least polar, and least specific interface in comparison to other IL/receptor complexes in the γc family. We map the mutations found in patients with SCID onto the IL-7Rα structure and show that they are localized outside the binding interface. Lastly, we discuss the possible mechanism of glycosylation of IL-7Rα with its binding interaction with IL-7. The binding kinetics and affinities observed for the interactions between IL-7 and unglycosylated or glycosylated IL-7Rα reveal the importance of glycosylation in formation of the IL-7/IL-7Rα complex. Surface plasmon resonance (SPR) binding kinetics collected on IL-7 from Escherichia coli (EC) and unglycosylated IL-7Rα (EC), glycosylated IL-7Rα from Chinese hamster ovary (CHO) cells, and glycosylated IL-7Rα from Schneider insect (S2) cells display biphasic kinetics that fit best to a two-state reaction mechanism with two on- and off-rate constants (Figure 1; Table 1). IL-7 binds to glycosylated IL-7Rα (CHO/S2) with k1 rates that are 7100-fold (CHO) and 5200-fold (S2) faster than the k1 rate of IL-7 binding to unglycosylated IL-7Rα (EC). The k2 rates of IL-7 binding to the IL-7Rα glycoforms (CHO/S2) display negligible effects in the comparison of IL-7 binding to unglycosylated IL-7Rα (4.9-fold for CHO to EC and 1.7-fold for S2 to EC). Similarly, the two off rates, k−1 and k−2, also show negligible effects in the comparison of IL-7 binding to glycosylated IL-7Rαs (CHO/S2) and unglycosylated IL-7Rα (7.1- and 2.4-fold for CHO to EC, and 5.9- and 3.4-fold for S2 to EC). Thus, the ∼300-fold enhancement in Kd of IL-7 and glycosylated IL-7Rα (CHO/S2) relative to IL-7 and unglycosylated IL-7Rα (EC) derives primarily from an accelerated k1 rate.Table 1Binding Constants of IL-7 to IL-7Rα Variantsk1 (M−1s−1)k−1 (s−1)k2 (s−1)k−2 (s−1)KdaKd = k−1k−2/k1(k2 + k−2).IC50bCompetition ELISA experiments were performed in PBS buffer (pH 7.4) with 0.05% Tween 20 at 25°C.wt-IL-7 (EC)IL-7Rα (EC)2.1 × 1021.7 × 10−24.2 × 10−31.2 × 10−318 μM3.0 μMIL-7Rα (CHO)1.5 × 1061.2 × 10−18.6 × 10−42.9 × 10−362 nMNDIL-7Rα (S2)1.1 × 1061.0 × 10−12.5 × 10−34.1 × 10−356 nM190 nMIL-7Rα (S2 Endo H)1.1 × 1061.1 × 10−11.4 × 10−36.2 × 10−382 nM340 nMIL-7Rα (S2 PNGase F)2.4 × 1038.3 × 10−23.5 × 10−32.3 × 10−314 μM2.2 μME106A-IL-7 (EC)IL-7Rα (S2)1.9 × 1051.7 × 10−23.2 × 10−31.0 × 10−321 nMNDIL-7Rα (S2 PNGase F)6.9 × 1036.9 × 10−26.0 × 10−39.8 × 10−41.4 μMNDND, not determined. Surface plasmon resonance experiments were performed in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20 at 25°C. Experiments were triplicated with standard errors < 10% for the rate constants.a Kd = k−1k−2/k1(k2 + k−2).b Competition ELISA experiments were performed in PBS buffer (pH 7.4) with 0.05% Tween 20 at 25°C. Open table in a new tab ND, not determined. Surface plasmon resonance experiments were performed in 10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.005% Tween 20 at 25°C. Experiments were triplicated with standard errors < 10% for the rate constants. The enhanced k1 and Kd of IL-7 to glycosylated IL-7Rα relative to unglycosylated IL-7Rα arises directly from N-glycosylation, but is insensitive to glycan composition. Binding studies of IL-7 with IL-7Rα (S2) treated with peptide:N-glycosidase F (PNGase F)—an enzyme that removes N-glycans and converts the Asns to Asps—resemble those performed on IL-7 and unglycosylated IL-7Rα (EC), indicating that the differences in binding result from N-glycosylation and not some attribute of using different cell expression systems (Figure 1D). The binding constants were also measured for IL-7 binding to glycosylated IL-7Rα expressed in two cell lines (CHO and S2) that modify proteins with different N-glycosylation patterns (Figures 1B and 1C). Unlike CHO cells, S2 cells do not incorporate complex branches with sialic acids or galactoses onto proteins, but express proteins with paucimannose hybrid glycans (Aoki et al., 2007Aoki K. Perlman M. Lim J.M. Cantu R. Wells L. Tiemeyer M. Dynamic developmental elaboration of N-linked glycan complexity in the Drosophila melanogaster embryo.J. Biol. Chem. 2007; 282: 9127-9142Crossref PubMed Scopus (208) Google Scholar). Even though the glycosylation patterns on IL-7Rα from S2 cells differ from those on IL-7Rα from CHO cells, the binding constants were comparable. Binding is likely unaffected by the variations in glycan branching because the proximal N-acetylglucosamine (GlcNAc) is wholly responsible for the large enhanced k1 on rate and affinity of IL-7 to glycosylated IL-7Rα. The binding constants measured for IL-7 to glycosylated IL-7Rα (S2) treated with endoglycosidase H (Endo H)—an enzyme that cleaves the bond between the first two GlcNAcs of hybrid mannose glycans like that produced from S2 cells, leaving the proximal GlcNAc—were similar to those measured for IL-7 to the fully glycosylated IL-7Rα (CHO/S2) (Figures 1B, 1C, and 1E). The SPR experiments measuring the affinities between IL-7 and unglycosylated IL-7Rα (EC) and glycosylated IL-7Rα (S2) untreated and treated with PNGase F or Endo H were further confirmed by competition ELISA experiments (Figure S2; Table 1). We determined the crystal structures of IL-7 (EC, E106A mutation) bound to both unglycosylated and glycosylated forms of the IL-7Rα at 2.7 and 2.9 Å resolution, respectively. Similar binding constants were observed between the interactions of E106A-IL-7 with both forms of IL-7Rα compared to wt-IL-7 (Table 1). The unglycosylated complex was refined with a crystallographic R value (Rcryst) of 0.212 (Rfree of 0.266; Table S1). Two unglycosylated IL-7/IL-7Rα (EC) complexes were located within the asymmetric unit. The glycosylated complex was refined to Rcryst and Rfree values of 0.234 and 0.267, respectively (Table S2). One glycosylated IL-7 (EC)/IL-7Rα (S2) complex was located within the asymmetric unit. IL-7 adopts an up-up-down-down four-helix bundle topology with two crossover loops (Figure 2A). The α helices A–D vary in length from 13 to 22 residues, similar to the lengths of helices in other γc ILs. A turn of a π helix from T12–M17 in helix A is unique to IL-7 and stabilizes the IL-7/IL-7Rα interaction. The first crossover loop of 23 residues contains 1.5 turns of α helix (mini-helix 1), whereas the majority of the second crossover loop of 33 residues could not be traced in any of the three complex structures. The first crossover loop in IL-7 is positioned differently between the IL-7/IL-7Rα complex structures, probably from local crystal packing contacts. The crystal structures of IL-7 clearly showed electron density to trace two out of three disulfide bonds (Figure 2A). The disulfide bonds are C34-C129, C47-C141, and C2-C92. This disulfide bond patterning is in contrast to homology models of IL-7 and biochemical data that predicted different locations by swapping the C47-C141 and C2-C92 disulfide bonds for C2-C141 and C92-C141 (Cosenza et al., 1997Cosenza L. Sweeney E. Murphy J.R. Disulfide bond assignment in human interleukin-7 by matrix-assisted laser desorption/ionization mass spectroscopy and site-directed cysteine to serine mutational analysis.J. Biol. Chem. 1997; 272: 32995-33000Crossref PubMed Scopus (27) Google Scholar, Cosenza et al., 2000Cosenza L. Rosenbach A. White J.V. Murphy J.R. Smith T. Comparative model building of interleukin-7 using interleukin-4 as a template: a structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation.Protein Sci. 2000; 9: 916-926Crossref PubMed Scopus (18) Google Scholar). Whereas the electron density allowed tracing of the C34-C129 and C47-C141 disulfide bonds, it was too weak near the N terminus (residues 1–6[7]) and the end of helix C proceeding into the second crossover loop (residues 89–123) to trace the third disulfide bond between C2 and C92. Similarly, electron density was absent for the N-terminal disulfide bond of a structure of IL-10 (Yoon et al., 2006Yoon S.I. Logsdon N.J. Sheikh F. Donnelly R.P. Walter M.R. Conformational changes mediate interleukin-10 receptor 2 (IL-10R2) binding to IL-10 and assembly of the signaling complex.J. Biol. Chem. 2006; 281: 35088-35096Crossref PubMed Scopus (89) Google Scholar). Previous studies indicated that W142 of helix D, the sole Trp of IL-7, was critical to the interaction between IL-7 and γc (Cosenza et al., 2000Cosenza L. Rosenbach A. White J.V. Murphy J.R. Smith T. Comparative model building of interleukin-7 using interleukin-4 as a template: a structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation.Protein Sci. 2000; 9: 916-926Crossref PubMed Scopus (18) Google Scholar, vanderSpek et al., 2002vanderSpek J.C. Sutherland J.A. Gill B.M. Gorgun G. Foss F.M. Murphy J.R. Structure function analysis of interleukin 7: requirement for an aromatic ring at position 143 of helix D.Cytokine. 2002; 17: 227-233Crossref PubMed Scopus (8) Google Scholar). However, the IL-7 structures show the burial of W142 into the hydrophobic core of the four-helix bundle, with the Nɛ forming a hydrogen bond (H bond) to T86 Oγ1 (Figure 2B). Thus, mutation of W142 likely causes defective folding of helix D and/or complete unfolding of IL-7, resulting in the reported decreases in cell assays (Cosenza et al., 2000Cosenza L. Rosenbach A. White J.V. Murphy J.R. Smith T. Comparative model building of interleukin-7 using interleukin-4 as a template: a structural hypothesis that displays atypical surface chemistry in helix D important for receptor activation.Protein Sci. 2000; 9: 916-926Crossref PubMed Scopus (18) Google Scholar, vanderSpek et al., 2002vanderSpek J.C. Sutherland J.A. Gill B.M. Gorgun G. Foss F.M. Murphy J.R. Structure function analysis of interleukin 7: requirement for an aromatic ring at position 143 of helix D.Cytokine. 2002; 17: 227-233Crossref PubMed Scopus (8) Google Scholar). IL-7Rα forms a L-shaped architecture similar to other IL receptors in the γc family, as well as other related receptors in the cytokine receptor class I (CRH I) superfamily, including receptors for growth hormone, erythropoietin, and other ILs (Figure 2C). The 219 residue IL-7Rα ectodomain folds into two fibronectin type III (FNIII) domains connected by a 310-helical linker. The elbow angle (ɛ) between the FNIII domains was similar in both IL-7Rα structures, ranging from 74° to 75° (Table S3). In the D1 domain of IL-7Rα, a disulfide bond (C22R-C37R) conserved among CRH I family members bridges β strands A1 and B1. Two other disulfide bonds unique to IL-7Rα connect β strands C1 to C′1 (C54R-C62R) and F1 to G1 (C88R-C98R). Another defining feature of the CRH I family found in IL-7Rα is the WSXWS sequence motif in the D2 domain. In IL-7Rα, the WSXWS motif forms extensive π cation side-chain stacking interactions critical to the SCID mutations discussed below. IL-7Rα contains six potential N-glycosylation sites: N29R, N45R, N131R, N162R, N212R, and N213R. The electron density of glycosylated IL-7Rα allowed tracing of two GlcNAcs for three different Asns: N29R, N45R, and N131R (Figures 2C–2E). The side chain of N162R was clear in the electron density, but not its potential glycan. Because electron density was absent for residues E210R-D219R, neither N212R nor N213R could be traced. All of the Asn side chains modeled in the glycosylated structure were also observable in the unglycosylated structures, except residue N29R, indicating extensive side-chain mobility. All of the glycans of the IL-7Rα structure are extending away from IL-7Rα, and none of the glycans are contacting other residues on IL-7Rα except the second GlcNAc (NAG 903) attached to N45R, whose O6 glycan group is H bonding to A48R N (3.2 Å). Although the overall structures of IL-7 bound to both forms of IL-7Rα are similar, a change in the position of the predicted γc binding interface suggests that glycosylation may also modulate the binding of γc to IL-7/IL-7Rα (Figure 3A). The two unglycosylated complexes superimpose with a root-mean-square deviation (rmsd) of 0.29 Å (164 Cα) for all secondary (2°) structural elements. Superimposing the 2° structural elements of the glycosylated structure onto either unglycosylated complex yield rmsds of 0.53 and 0.64 Å (159 Cα), demonstrating that the glycans do not induce large conformational changes. Independently, the IL-7 four-helix bundles superimpose with an rmsd of 0.52 Å (72 Cα, glycosylated structure to both unglycosylated structures), and the two IL-7Rα FNIII domains superimpose with an rmsd of 0.37 Å (86 Cα, glycosylated structure to both unglycosylated structures). The surface on IL-7 where γc is predicted to bind (helices A and D) experienced the largest structural change between the unglycosylated and glycosylated structures (Figure 3A). When the complex structures are aligned on the IL-7Rαs using both D1 and D2 domains, the IL-7 four-helix bundles shift relative to one another with a displacement of individual helices ranging from 1° (helix B) to 5° (helix D) (Figure 3A). If the complex structures are aligned on the IL-7 four-helix bundles, then the D2 domains of the α receptors rotate accordingly. Similar displacements are observed between the glycosylated complex with both unglycosylated complexes, potentially ruling out changes induced by crystal contact formation. Thus, glycosylation of IL-7Rα may be positioning the four-helix bundle of IL-7 and/or the domains of the IL-7Rα in a manner conducive for γc binding. Further studies will probe whether glycosylation of IL-7Rα modulates the binding constants, structure, and function of γc. IL-7 is positioned at the elbow region connecting the D1 and D2 domains of IL-7Rα (Figures 2C, 2D, and 3A). The interface largely comprises hydrophobic and van der Waals (VDW) interactions, although a few intermolecular H bonds exist in the binding interface (Figures 3B–3E; Table 2). Residues in helices A and C of IL-7 contact residues in the six loop regions connecting the β sheets of the IL-7Rα FNIII domains (Figures 3C and 3D). On average, apolar residues dominate the contact interfaces for the IL-7/IL-7Rα structures over polar residues (47% versus 33%; Table 3). The two unglycosylated complexes in the asymmetric unit bury 735 and 720 Å2 of surface area, whereas the glycosylated complex buries 705 Å2 of surface area (Table 3).Table 2Potential Hydrogen Bonds in Unglycosylated and Glycosylated IL-7/IL-7Rα ComplexesUnglycosylated IL-7/IL-7Rα Complex 1IL-7IL-7RαDistance (Å)Asp74 Oδ1Lys77 Nζ3.4Asp74 Oδ1Ser31 Oγ3.2Asp74 Oδ2Lys77 Nζ3.1Lys81 NɛLys77 O3.1Gln11 Nɛ2Tyr192 Oη3.1Wat-1Ser19 (IL-7) Oγ2.7Wat-1Tyr139 (IL-7Rα) Oη2.6Wat-15Glu84 (IL-7) Oɛ13.5Wat-15Glu84 (IL-7) Oɛ23.6Wat-15Val58 (IL-7Rα) O2.6Wat-15Val60 (IL-7Rα) O3.7Unglycosylated IL-7/IL-7Rα Complex 2Asp74 Oδ1Lys77 Nζ3.4Asp74 Oδ1Ser31 Oγ3.3Asp74 Oδ2Lys77 Nζ3.1Lys81 NζLys77 O2.9Gln11 Nɛ2Tyr192 Oη3.2Glycosylated IL-7/IL-7Rα ComplexIL-7IL-7RαGln22 Oɛ1Lys138 Nζ3.4Asp74 Oδ2Ser31 Oγ3.3Asp74 Oδ2Lys77 Nζ2.9Asp74 Oδ1Lys77 Nζ3.6Lys81 NζLys77 O2.7 Open table in a new tab Table 3Summary of Binding Interfaces of γc ComplexesComplexBSA (Å2)aAverage buried surface area of the cytokine and receptor at the interface.H BondsScbShape complementarity of the interface.% Polar Residues% Apolar ResiduesUnglyco IL-7/IL-7Rα 173550.6829.751.5Unglyco IL-7/IL-7Rα 272050.6933.147.2Glyco IL7/IL-7Rα70550.6536.342.5IL-2/IL-2Rβ135080.7439.324.1IL-4/IL-4 binary835150.7439.730.8IL-4/IL-4Rα ternary778140.7046.022.2Values were calculated from PISA, CCP4, and the protein/protein interaction server at http://www.bioinformatics.sussex.ac.uk/protorp/index.html.a Average buried surface area of the cytokine and receptor at the interface.b Shape complementarity of the interface. Open table in a new tab Values were calculated from PISA, CCP4, and the protein/protein interaction server at http://www.bioinformatics.sussex.ac.uk/protorp/index.html. Glycosylation of IL-7Rα does not influence its interaction with IL-7 through direct contacts at the binding interface with IL-7 (Figure 2D). The glycans attached to N45R face away from IL-7 on the back side of the D1 domain of IL-7Rα. The glycans attached to N29R and N131R are 10 and 15 Å away from the closest IL-7 atom, respectively. Even though N162R, N212R, and N213R could not be visualized in the electron density, their potentially attached glycans are not near the interface either. Ongoing biophysical and structural studies of the free states of unglycosylated and glycosylated IL-7 and IL-7Rα will determine how glycosylation affects molecular recognition events of IL-7Rα with IL-7. The IL-7/IL-7Rα interfaces in all three structures involve similar H bonds, except for a couple of bonds and water-mediated interactions. The binding interfaces of both complexes share four common H bonds involving D74 and K81 of IL-7, but each complex has additional H bonds absent from the other complex (Figure 3E; Table 2). In the glycosylated complex, Q22 Oɛ1 from helix A forms a long H bond with K138R Nζ (3.4 Å), whereas, in the unglycosylated complex, the Y192R Oη forms an H bond with Q11 Nɛ2 (3.2 Å). Only one of the unglycosylated complexes in the asymmetric unit has an interface containing water-mediated interactions. Of the six water-mediated interactions observed in the unglycosylated structure, two H bonds form between the hydroxyl side chains of S19 of helix A and Y139R, and four H bonds form between the side chain of E84 of helix C and the backbone carbonyls of V58R and V60R in loop CC′1 of IL-7Rα (Figure 3E). The differences observed in the H bonds among the three structures involve rotameric changes of the side chains requiring minimal energetic costs going from one structure to another. IL-7/IL-7Rα displays a more complex binding mechanism in comparison to other IL/receptor interactions in the γc family. The binding kinetics of IL-7 with either unglycosylated or glycosylated IL-7Rα involve a two-step reaction where an encounter complex, (IL-7:IL-7Rα)∗, is observed before reaching the final complex state (k−1 > k2; Equation 1). In contrast, the SPR binding kinetics of IL-2, -4, and -21 to their CRH I receptors were fit best to a single-step reaction with a single on- and off-rate constant (k1, k−1) (Liparoto et al., 2002Liparoto S.F. Myszka D.G. Wu Z. Goldstein B. Laue T.M. Ciardelli T.L. Analysis of the role of the interleukin-2 receptor γ chain in ligand binding.Biochemistry. 2002; 41: 2543-2551Crossref PubMed Scopus (36) Google Scholar, Shen et al., 1996Shen B.J. Hage T. Sebald W. Global and local determinants for the kinetics of interleukin-4/interleukin-4 receptor α chain interaction. A biosensor study employing recombinant interleukin-4-binding protein.Eur. J. Biochem. 1996; 240: 252-261Crossref PubMed Scopus (74) Google Scholar, Zhang et al., 2003Zhang J.L. Foster D. Sebald W. Human IL-21 and IL-4 bind to partially overlapping epitopes of common γ-chain.Biochem. Biophys. Res. Commun. 2003; 300: 291-296Crossref PubMed Scopus (29) Google Scholar). An encounter complex of these complexes may exist, but not observable using current methods. The significance of the two-step binding reaction of the human IL-7/IL-7Rα interaction and the dramatic enhancement of the k1 on rate of IL-7 to glycosylated IL-7Rα are open questions that future studies will explore. Of note, the interaction between mouse IL-7 and full-length mouse IL-7Rα on a cell surface also displayed biphasic binding kinetics (Park et al., 1990Park L.S. Friend D.J. Schmierer A.E. Dower S.K. Namen A.E. Murine interleukin 7 (IL-7) receptor. Characterization on an IL-7-dependent cell line.J. Exp. Med. 1990; 171: 1073-1089Crossref PubMed Scopus (127) Google Scholar). The point mutations in IL-7Rα that have been identified in patients suffering from SCID map to residues outside the binding epitope with IL-7, but localize to residues in the hydrophobic cores of the FNIII domains, cysteines of disulfide bonds, or the WSXWS motif (Figure 4; reviewed in Giliani et al., 2005Giliani S. Mori L. de Saint Basile G. Le Deist F. Rodriguez-Perez C. Forino C. Mazzolari E. Dupuis S. Elhasid R. Kessel A. et al.Interleukin-7 receptor α (IL-7Rα) deficiency: cellular and molecular bases. Analysis of clinical, immunological, and molecular features in 16 novel patients.Immunol. Rev. 2005; 203: 110-126Crossref PubMed Scopus (133) Google Scholar). Five SCID mutations identified in the IL-7Rα D1 domain include G8RR, S24RR, L35RR, C54RY, and C98RY. The G8RR mutation could not be mapped onto the IL-7Rα structure because residue G8 was not visible in the electron density of any of the IL-7Rα structures. Ser24R Oγ forms a H bond to L130R O that will be absent from the S24RR SCID mutation. The loss of this H bond and the need to accommodate a large Arg side chain probably causes the S24RR mutation to destabilize the linker connecting the D1 and D2 domains. The L35RR SCID mutation presumably unfolds the FNIII domain by forcing a bulky, polar side chain into the hydrophobic core of the D1 domain. SCID mutations C54RY and C98RY each remove a disulfide bond from IL-7Rα, thereby disrupting the folding/stability of the D1 domain. SCID mutations in the D2 domain localize to or near the WSXWS motif between W197R and S201R. The Trps, W197R and W200R, of the WSXWS motif participate in extensive π cation interactions with Trp (W158R), Lys (K184R), and arginine (R186R and R150R) side chains. Two SCID mutations convert what would be residues W197R and R186R to stop codons, resulting in termination of mRNA. Three other SCID mutations in the WSXWS motif include S198RN, W200RC, and S201RI. S198R and S201R rigidify the D2 domain by orienting the G2 and F2 β strands through several Ser side-chain to main-chain H bonds. The SCID mutations S198RN and S201RI lack these critical H bonds. In addition to disrupting the core of the π cation interactions, W200RC may interfere with proper disulfide bond formation. Finally, SCID mutations L115RR, P112RH, and P112RS probably destabilize the hydrophobic core of the D2 domain. Studies of related CRH I cytokine receptors (GHR and EpoR) reported the WSXWS motif as crucial to proper folding of the ectodomains, binding of ligands, and functions (Baumgartner et al., 1994Baumgartner J.W. Wells C.A. Chen C.M. Waters M.J. The role of the WSXWS equivalent motif in growth hormone receptor function.J. Biol. Chem. 1994; 269: 29094-29101Abstract Full Text PDF PubMed Google Scholar, Hilton et al., 1996Hilton D.J. Watowich S.S. Katz L. Lodish H.F. Saturation mutagenesis of the WSXWS motif of the erythropoietin receptor.J. Biol. Chem. 1996; 271: 4699-4708Abstract Full Text Full Text PD" @default.
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