Báo cáo khoa học: Type I receptor binding of bone morphogenetic protein 6 is dependent on N-glycosylation of the ligand pdf

Type I receptor binding of bone morphogenetic protein is dependent on N-glycosylation of the ligand Stefan Saremba1,2,*, Joachim Nickel1,*, Axel Seher1, Alexander Kotzsch1,2, Walter Sebald1 and Thomas D Mueller1,2 Lehrstuhl fur Physiologische Chemie II, Biozentrum der Universita Wurzburg, Germany ăt ă ă Lehrstuhl fur Molekulare Panzenphysiologie und Biophysik, Julius-von-Sachs Institut der Universitat Wurzburg, Germany ă ă ă Keywords crystal structure; ligand–receptor specificity; protein–protein interaction; recognition; transforming growth factor-b superfamily Correspondence T D Mueller, Lehrstuhl fur Molekulare ă Panzenphysiologie und Biophysik, Juliusvon-Sachs Institut der Universitat Wurzburg, ă ă Julius-von-Sachs Platz 2, D-97082 Wurzburg, Germany ă Fax: +49 931 888 6158 Tel: +49 931 888 6146 E-mail: mueller@biozentrum uni-wuerzburg.de Website: http://www.bot1.biozentrum uni-wuerzburg.de *These authors contributed equally Database The coordinates and structure factors for the structures of wild-type BMP-6 and B2BMP-6 have been deposited with the Protein Data Bank, entry codes 2R52 and 2R53 Bone morphogenetic proteins (BMPs), together with transforming growth factor (TGF)-b and activins ⁄ inhibins, constitute the TGF-b superfamily of ligands This superfamily is formed by more than 30 structurally related secreted proteins The crystal structure of human BMP-6 was determined ˚ to a resolution of 2.1 A; the overall structure is similar to that of other TGF-b superfamily ligands, e.g BMP-7 The asymmetric unit contains the full dimeric BMP-6, indicating possible asymmetry between the two monomeric subunits Indeed, the conformation of several loops differs between both monomers In particular, the prehelix loop, which plays a crucial role in the type I receptor interactions of BMP-2, adopts two rather different conformations in BMP-6, indicating possible dynamic flexibility of the prehelix loop in its unbound conformation Flexibility of this loop segment has been discussed as an important feature required for promiscuous binding of different type I receptors to BMPs Further studies investigating the interaction of BMP-6 with different ectodomains of type I receptors revealed that N-glycosylation at Asn73 of BMP-6 in the wrist epitope is crucial for recognition by the activin receptor type I In the absence of the carbohydrate moiety, activin receptor type I-mediated signaling of BMP-6 is totally diminished Thus, flexibility within the binding epitope of BMP-6 and an unusual recognition motif, i.e an N-glycosylation motif, possibly play an important role in type I receptor specificity of BMP-6 (Received September 2007, revised November 2007, accepted 12 November 2007) doi:10.1111/j.1742-4658.2007.06187.x Bone morphogenetic protein (BMP)-6, BMP-5, BMP-7 and BMP-8 constitute a subgroup of the transforming growth factor (TGF)-b superfamily proteins Besides the ability of BMP-6 to induce bone formation at ectopic and orthotopic sites, BMP-6 transcripts have been localized in numerous studies to developing organs and tissues, such as the heart, the brain, and hyper- trophic cartilage, throughout the developing skeletal system, and also to adult tissues, such as brain and uterus [1–5] BMP-6 and its closest relative, BMP-7, show overlapping expression patterns as well as overlapping functions For example, in the developing heart, BMP-6 and BMP-7 are required for cushion formation and septation [5] In the brain, BMP-6 and Abbreviations ActR, activin receptor; ALP, alkaline phosphatase; BMP, bone morphogenetic protein; BMPR, bone morphogenetic protein receptor; GlcNAc, N-acetylglucosamine; h, human; MPD, 2-methyl-2,4-pentanediol; TGF, transforming growth factor 172 FEBS Journal 275 (2008) 172–183 ª 2007 The Authors Journal compilation ª 2007 FEBS S Saremba et al BMP-7 have important effects at early and late stages of nervous system development (e.g specification of nervous system patterning [6] and decision of neuronal fate [7]) BMP-6-deficient mice are viable and fertile without displaying overt effects in tissues known to express BMP-6 mRNA [3], implying also a functional redundancy among the factors of this subgroup BMPs exert their biological effects by inducing the formation of a heteromeric receptor complex from type II [activin receptor (ActR)-II, ActR-IIB, or BMP receptor (BMPR)-II] and type I chains (BMPR-IA, BMPR-IB, and ActR-I) [8–10] The constitutively active type II kinase then phosphorylates and thereby activates the type I chain [11], which subsequently propagates the signal downstream by acting on BMPR-regulated Smads-1 ⁄ ⁄ [12] Although BMPs are characterized by versatility in receptor binding, referred to as promiscuity, tremendous differences underlie in vivo signaling by either BMP-2 ⁄ or BMP6 ⁄ 7: whereas BMP-2 ⁄ exert their function by initial binding to the high-affinity receptors BMPR-IA or BMPR-IB, it has been shown that ActR-I is the predominant type I receptor used by BMP-7 in a variety of cell lines [13] The overall fold and dimer architecture seem to be highly conserved for the ligands of the TGF-b superfamily, when the receptor unbound conformation is considered Several structures have been determined, i.e TGF-b1, TGF-b2, TGF-b3, BMP-2, BMP-7, and growth and differentiation factor (GDF)-5, all showing the canonical fold Here, we present the crystal structure of BMP-6 and compare the structure with that of other BMPs Differences resulting in possible alterations of the type I receptor-binding profile were investigated Comparison of the receptor binding of Escherichia coli and CHO-cell derived BMP-6 reveals the importance of N-glycosylation for ActR-I binding and activation For proteins other than TGF-b ligands (e.g hormones), it has been shown that glycosylation can ameliorate the receptor binding [14]; however, this is the first report in which glycosylation of a TGF-b superfamily ligand is essential for the versatility in receptor binding Results and Discussion Structure of human BMP-6 Structures of BMP-6 and B2-BMP-6 were determined by molecular replacement using the coordinates of human (h)BMP-7 (Protein Data Bank entry 1BMP [15]) as a start model Analysis of the unit cell content suggested the presence of a complete BMP-6 dimer in Type I receptor specificity of BMP-6 the asymmetric unit Assuming the presence of the BMP-6 dimer in the asymmetric unit, the Matthews ˚ coefficient VM is 3.94 A3ỈDa)1, corresponding to a solvent content of  70% With only one monomer present in the asymmetric unit, the solvent content would exceed 86%, making this possibility unlikely Calculation of a self-rotation function confirmed the presence of a two-fold noncrystallographic symmetry, which is distinct from all other structures of members of the TGF-b superfamily that have so far been determined (e.g BMP-2 [16], BMP-7 [15], GDF-5 [17,18], TGF-b2 [19,20], and TGF-b3 [21]) As a result, the biological dimer is not formed by a crystallographic dyad running through the intermolecular disulfide bond, as is observed in all the other structures of TGF-b members, but by a noncrystallographic two-fold axis, indicating possible asymmetry in the homodimeric structure (Fig 1A,B) Indeed, our structure data imply that loop regions, e.g the prehelix loop, in BMP-6 can adopt two different conformations (Fig 1C) Superimposing the structure of free BMP-2 [16] onto the two different monomer conformers of BMP-6 yields rmsd values of ˚ 2.5 and 1.4 A for the Ca positions, clearly indicating that one of the two BMP-6 conformers adopts a loop conformation similar to that of BMP-2 (Fig 1D) If only the Ca atoms of the b-sheet core of the BMP-2 dimer are considered (without helix, fingertip and pre˚ ˚ helix loops), an rmsd of A (0.8 and 0.6 A for the individual monomer subunits) is observed, showing that the core b-sheet and the dimer architecture are almost identical between BMP-6 and BMP-2 (Fig 1D) The region exhibiting the largest difference between the two monomers is the prehelix loop comprising residues Phe66 to Met72 of BMP-6 (Fig 1C) In conformer 1, the loop strongly deviates from the canonical backbone conformation observed in all other BMP (see Fig 1E,F for BMP-2 as an example) members [22] As compared with BMP-2, distances between the Ca atoms of individual residues of up to more than ˚ A, e.g between His71 (BMP-6) and His54 (BMP-2), ˚ are found The smallest distance (1.8 A) between two Ca atoms of BMP-6 and BMP-2 within this loop segment is observed for Leu68 (BMP-6) and Leu51 (BMP-2) (Fig 1E,F) The prehelix loop, however, was shown to contain the main binding and specificity determinants for type I receptor recognition in BMP-2 [23] and GDF-5 [17] Structure analysis of receptor–ligand complexes of BMP-2 [23,24] and BMP-7 [25] suggested that receptor binding and recognition is accompanied by an induced fit mechanism affecting the side chain and backbone conformation in this loop region Thus, the fact that FEBS Journal 275 (2008) 172–183 ª 2007 The Authors Journal compilation ª 2007 FEBS 173 Type I receptor specificity of BMP-6 A S Saremba et al B Fig The prehelix loop of BMP-6 adopts two conformations (A) Ribbon representation of BMP-6 viewed from the top and from the side (B) The central intermolecular disulfide bond is indicated by ball-and-stick, secondary structure elements, and structural features are marked The prehelix loop adopts two vastly different conformations, with the largest distances between the Ca atoms of the same amino acid residue in both segments (C) Stereoview of a superposition of the prehelix loop of conformers A and B of BMP-6 Residues occupying similar positions are indicated in black; residues having different orientations in the two conformers are marked A and B according to the conformer (D) Stereoview of a superposition of BMP-6 and BMP-2 (Protein Data Bank entry 3BMP), showing the differences in the loop conformations of the fingertip loops as well as the prehelix loop (E) Superposition of the prehelix loops of BMP-2 (red carbon atoms) and BMP-6 (cyan carbon atoms) in its canonical loop conformation, which is very similar to that of BMP-2 (F) Same as in (E) except for the BMP-6 prehelix loop of conformer (green carbon atoms), which adopts a noncanonical conformation, and is therefore different from BMP-2 C D E F BMP-6 shows two possible, largely different conformations for this loop segment suggests that recognition and binding of type I receptors might be influenced by this unique feature (Fig 1C) Superimposing the structure of the binary complex of BMP-2 bound to its high-affinity receptor BMPR-IA indeed shows that the noncanonical conformation of the prehelix loop of BMP-6 would prevent binding of BMPR-IA, due to steric hindrance, whereas the canonical loop conformation (BMP-2-like conformation) could form similar noncovalent interactions with the type I receptor (Fig 2) Although the noncanonical loop conformation of BMP-6 seems not to be able to form a stable ligand–receptor interface using the type I receptor structures known so far, the two different loop conformations clearly show that the prehelix loop seems to be dynamically disordered in the unbound ligand Together with the fact that the a-helix of the type I 174 receptor BMPR-IA, which carries the main binding determinants for BMP-2 interaction (Phe85 and Gln86 of BMPR-IA), also seems not to be folded in the free receptor [26], a large portion of the core interface seems to be flexible and undergoes a disorder-to-order transition upon complex formation This induced-fit mechanism might explain the high degree of promiscuity in the BMP ligand–receptor interaction, as it allows the ligand as well as the receptor surfaces to adapt to the binding partner Receptor binding and activity of BMP-6 depend on the nature of the expression system Signaling of BMP-6 and BMP-7 has been shown to be mediated mainly via the ActR-I receptor in many cell types [9,13,27], although this receptor binds both BMPs only with weak affinities [25] In contrast, FEBS Journal 275 (2008) 172–183 ª 2007 The Authors Journal compilation ª 2007 FEBS S Saremba et al Type I receptor specificity of BMP-6 A Fig Analysis of a BMP-6–BMPR-IA complex model (A) A putative model of BMP-6 bound to BMPR-IA was built by docking the BMPR-IA molecules of the BMP-2–BMPR-IA complex (Protein Data Bank entry 1REW) to BMP-6 (B) The noncanonical prehelix loop is incompatible with complex formation, due to several steric clashes between residues of the BMP-6 prehelix loop and the b5a1-loop of BMPR-IA (C) In its canonical (or BMP-2 like) form, the prehelix loop adopts a conformation that is very similar to that of BMP-2 in the BMP-2–BMPR-IA complex No severe steric clashes are found, suggesting that this loop conformation might be adopted in a BMP-6–BMPR-IA interaction B BMP-2 uses the type I receptors BMPR-IA and BMPR-IB, both of which are bound with high affinities To further elucidate the molecular basis for the different type I receptor specificity profiles of BMP-2, BMP-6, and BMP-7, we used in vitro interaction analysis (Table 1) Ligand proteins of BMP-2 (E coli), BMP-6 (CHO cells) and BMP-7 (NS0 cells) were immobilized onto a biosensor chip, and interaction with the receptor ectodomain proteins BMPR-IA, BMPR-IB and ActR-I was measured using surface plasmon resonance spectroscopy (BIAcore technique) As expected, immobilized BMP-2 showed high binding affinities for BMPR-IA and BMPR-IB (KD = 10 and 95 nm, respectively), whereas binding to ActR-I was below the detection level (KD > 400 lm) In contrast, BMP-7 bound to BMPR-IA with a much lower affinity of  10 lm and to BMPR-IB with a slightly higher affinity of about lm Binding of BMP-7 to ActR-I yielded affinities (KD  50 lm) similar to those described by Greenwald et al [25], and CHO cellderived BMP-6 showed a receptor binding profile similar to that of BMP-7 But, to our surprise, BMP-6 expressed from E coli did not bind to ActR-I (Table 2), whereas basically identical binding parameters for the type I receptors BMPR-IA and BMPR-IB and for the type II receptor ActR-II were observed (Table 2) Therefore, it can be ruled out that the difference in binding is caused by misfolding or unfolding of the E coli-derived BMP-6 We thus investigated whether E coli-derived BMP-6, which does not bind C Table Receptor binding profile of BMP-6, BMP-7, and BMP-2 (BIAcore analysis) Biosensor analysis using surface plasmon resonance was performed to determine binding affinities of the BMP ligand–receptor interaction Ligands were immobilized onto the surface of a CM5 sensor chip, and receptor ectodomain proteins were used as analyte Thus, interaction analysis yields the : interaction of BMPs and their receptor ectodomain proteins NB, no binding within detection limit (upper limit: KD > 400 lM) Ligands [affinity (lM)]a Receptor proteins Type I BMPR-IA BMPR-IB ActR-I Type II ActR-II BMP-6 BMP-7 BMP-2 1.6 0.39 39 10 1.1 55 0.015b 0.095b NB 4.5 0.9 3.8 a KD(eq) as deduced from the dose dependency of equilibrium binding b KD(kin) as deduced from the association and dissociation rates of the interaction; analysis of dose dependency of equilibrium binding yields higher (three-fold) values for KD(eq), as real equilibrium binding cannot be achieved, due to the slow association rates (kon  · 104ỈM)1Ỉs)1) of the BMP-2–BMPR-IA and BMPR-IB : interaction In contrast, association, and especially dissociation, for interaction of BMPs with ActR-I and ActR-II are faster (kon > 105ỈM)1Ỉs)1, koff > 10)1Ỉs)1), impeding the analysis of the dissociation rate and thus requiring analysis of the equilibrium binding ActR-I, is inactive in cell-based assays, as would be expected if ActR-I were the main signaling receptor for BMP-6 and BMP-7 Indeed, glycosylated BMP-6 and BMP-7 induced alkaline phosphatase (ALP) FEBS Journal 275 (2008) 172–183 ª 2007 The Authors Journal compilation ª 2007 FEBS 175 Type I receptor specificity of BMP-6 S Saremba et al Table N-glycosylation of BMP-6 is required for ActR-I binding (BIAcore analysis) Biosensor analysis using surface plasmon resonance was performed to determine binding affinities of the BMP-6 ligand–receptor interaction BMP-6 proteins were immobilized onto the surface of a CM5 sensor chip via amino-coupling, and receptor ectodomain proteins were used as analyte Thus, interaction analysis yields the : interaction of BMPs and their receptor ectodomain proteins NB, no binding within detection limit (upper limit: KD > 400 lM) Ligands [affinity (lM)a] BMP-6 (CHO) Receptor proteins Type I BMPR-IA BMPR-IB ActR-I Type II ActR-II BMP-6 (CHO) PNGase F BMP-6 (CHO) PNGase F3 ⁄ H BMP-6 (E coli) 1.7 0.37 27 2.2 0.45 NB 1.9 0.53 49 1.8 0.41 NB 4.9 4.4 5.4 5.3 a KD(eq) as deduced from the dose dependency of equilibrium binding expression in ATDC-5 cells in a dose-dependent manner, with EC50 values of and 57 nm (Fig 3A) In contrast, BMP-6 derived from E coli was practically inactive at ALP induction, even at the high concentrations tested (Fig 3A) It is also interesting to note that BMPR-IA seems to be not able to rescue activity of BMP-6 in ATDC5 cells, despite the fact that in vitro interaction analysis shows that BMP-6 (and BMP-7) can bind to BMPR-IA and BMPR-IB with higher affinity than ActR-I That signaling of BMP-6 and To determine the molecular basis for these differences between BMP-6 derived from E coli or CHO cells, we investigated whether post-translational modifications might play a role in receptor binding and activity The crystal structure analysis of recombinant BMP-7 expressed in CHO cells (Protein Data Bank entry 1LXI [25]) or complexes of BMP-7 (Protein Data Bank entries 1LX5 [25] and 1M4U [30]) showed that the N-glycosylation sequence Asn-X-Ser ⁄ Thr in the cystineknot motif, which is conserved among BMP ligands of the BMP-2 ⁄ and the BMP-5 ⁄ ⁄ family, does indeed B 2.5 2.0 Recognition of BMP-6 by ActR-I depends on N-glycosylation [3H]-thymidine incorporation (cpm) ALP activity (E405 nm) A BMP-7 is mediated via ActR-I can be seen from the inhibition of proliferation in the human myeloma cell line INA6, which expresses ActR-I but not BMPR-IA or BMPR-IB [28] Both BMP-6 and BMP-7 showed high activity in this cell line, whereas BMP-2, which signals via BMPR-IA and BMPR-IB, did not (Fig 3B) This is the first time that such a large difference in binding and activity has been observed for members of the TGF-b superfamily For example, BMP-2 derived from either prokaryotic or eukaryotic expression systems has similar biological activities when tested in various cells, e.g ALP induction in C2C12 or ATDC5 cells (W Sebald, unpublished results) Receptor binding is only marginally influenced, with a slightly decreased affinity of CHO cellderived BMP-2 for the type II receptor ActR-IIB [29] BMP-2 (5.3 nM+/–0.3) BMP-6 (9.3 nM+/–0.5) BMP-7 (56.7 nM+/–3.4) BMP-6 E.coli 1.5 1.0 0.5 0.0 10 BMP variant (nM) 100 14 000 12 000 10 000 8000 6000 4000 2000 BMP-2 (n.d.) BMP-6 (12.4 nM +/–0.8) BMP-7 (24.0 nM +/–2.2) BMP-6 E.coli (n.d.) control 10 100 BMP variant (nM) Fig Biological activities of BMP-6, BMP-7, and BMP-2 (A) Induction of ALP expression in ATDC5 cells is stimulated by BMP-6, BMP-7, and BMP-2 BMP-6 derived from E coli expression is inactive in these cells, probably due to its lack of binding to ActR-I (B) Signaling of BMP-6 and BMP-7 via ActR-I is shown in the inhibition of proliferation in the myeloma cell line INA6, which lacks the type I receptors BMPR-IA and BMPR-IB Whereas BMP-6 and BMP-7 show high activity in this cell line, BMP-2 is almost completely inactive, due to its requirement for BMPR-IA or BMPR-IB E coli-derived BMP-6 is also inactive, due to its inability to bind ActR-I; the green dashed line indicates maximal proliferation in the absence of any BMP ligand 176 FEBS Journal 275 (2008) 172–183 ª 2007 The Authors Journal compilation ª 2007 FEBS S Saremba et al Type I receptor specificity of BMP-6 carry carbohydrate moieties The two putative N-glycosylation sites in the N-terminus of the mature part of BMP-7, which are also present in BMP-6, have been shown not to be glycosylated [31,32] The binding site of the type I receptors is located in the so-called wrist epitope of BMPs, which comprises a part of both fingers and the prehelix loop, suggesting that the carbohydrate moieties linked to Asn73 of BMP-6 (Asn80 on BMP-7) could contact the type I receptors and thus modulate receptor binding To confirm this hypothesis, we performed deglycosylation of CHO cell-expressed BMP-6 and determined its receptor-binding properties by BIAcore interaction analysis First, we removed all N-linked carbohydrate by N-endoglycosidase F treatment under nondenaturing conditions to ensure that the folding of BMP-6 was not altered by the enzymatic reaction Endoglycosidase F hydrolyzes the N-glycosidic bond between the asparagine and the first N-acetylglucosamine (GlcNAc) residue, resulting in a nonglycosylated protein (Fig 4A) The completeness of the deglycosylation was checked by SDS ⁄ PAGE (Fig 4B) and MS analysis; the BMP-6 was then immobilized onto a biosensor, and the properties of binding to BMP type I and type II receptors were determined by BIAcore analysis Whereas binding to the type I receptors BMPR-IA and BMPR-IB, as well as to the type II receptor ActR-II, was essentially identical to binding of fully glycosylated BMP-6, no binding to A PNGase F ActR-I could be determined (Table 2) This clearly shows that binding of BMP-6 to ActR-I requires carbohydrate moieties attached to Asn73 as binding determinants, whereas the other type I receptors not As the parameters for binding to BMPR-IA, BMPR-IB and ActR-II are not influenced by the removal of the N-glycosylation, large, and even small, local structural changes can be excluded We examined how many carbohydrate residues might be involved in the binding of ActR-I by using a mixture of N-endoglycosidase H and N-endoglycosidase F3 The latter cleaves the b1–4 glycosidic bond between the first and the second GlcNAc residue, leaving the first carbohydrate (GlcNAc) attached to the protein (Fig 4A,B) Measurement of the binding affinities of this partially glycosylated BMP-6 for BMPR-IA and BMPR-IB confirms that binding to these two type I receptors is not altered by different N-glycosylation levels However, binding affinity for ActR-I is now very close (less than a factor of 2) to that of CHO cell-derived BMP-6 with full N-glycosylation (Table 2), showing that the first carbohydrate moiety at Asn73 is a main binding determinant for ActR-I interaction, whereas further carbohydrate residues in the carbohydrate chain are not required The aspartyl side chain generated from Asn by deglycosylation using endoglycosidase F cannot be responsible for this lack of activity, as the unglycosylated E coli BMP-6 containing an Asn at position 73 is also inactive B R1 GlcN M GlcN M β1-4 35 R2 25 18.4 M R1 α1-6 GlcN GlcN 18.4 14.4 β1-4 M β1-2 α1-4 α1-3 PNGases 14.4 GlcN Asp M GlcN 25 PNGase F3/H GlcN β1-2 45 35 Asp R 45 β1-4 R2 Fig Deglycosylation of BMP-6 expressed from CHO cells (A) Scheme to illustrate the restriction sites for the endoglycosidases used PNGase F hydrolyzes the N-glycosidic bond immediately after the asparagine residue, leaving a fully deglycosylated protein A mixture of PNGase F3 and PNGase H is used to trim complex carbohydrate structures to a single GlcNAc moiety attached to the asparagine residue (B) SDS ⁄ PAGE analysis under reducing conditions of the deglycosylation reactions of BMP-6, showing the completeness of the enzymatic reactions M: molecular weight marker Lane 1: BMP-6 derived from CHO cells Lane 2: BMP-6 after PNGase F treatment Lane 3: BMP-6 before PNGase F3 ⁄ H treatment Lane 4: BMP-6 after PNGase F3 ⁄ H treatment BMP-6 runs as two bands, i.e dimer and monomer FEBS Journal 275 (2008) 172–183 ª 2007 The Authors Journal compilation ª 2007 FEBS 177 Type I receptor specificity of BMP-6 S Saremba et al Modeling the interaction of BMP-6 and ActR-I To gain insights into how ActR-I might interact with the N-glycosylation site at Asn73 of BMP-6, we constructed a model of the binary complex of BMP-6 bound to ActR-I on the basis of our BMP-6 structure and the structures of the BMP-7–ActR-II (Protein Data Bank entry 1LX5 [25]) and BMP-2–BMPR-IA (Protein Data Bank entry 1REW [23]) complexes A putative carbohydrate chain was added to BMP-6 by using the complex glycosylation structure present on BMP-7 as identified in BMP-7–ActR-II and which also presents a typical N-glycosylation from expression in mammalian cells The model of the extracellular domain of ActR-I is based on the structure of BMPRIA in its bound conformation to BMP-2 (Protein Data Bank entry 1REW); insertions and deletions were built manually using quanta2006 software The putative complex model of BMP-6(glycosylated)–ActR-I was then formed by superimposing BMP-6(glycosylated) and ActR-I with the ligand and receptor structures in BMP-2–BMPR-IA (Protein Data Bank entry 1REW) The model of BMP-6–ActR-I shows that several residues in the N-terminus, the b1b2-loop and the short loop before the a-helix of ActR-I are in close proxim- ity to the carbohydrate chain of the BMP-6(glycosylated) model (Fig 5A,B) Residues of ActR-I in these regions, namely Lys11 and Tyr54, can possibly form several hydrogen bonds with the first carbohydrate moiety (Fig 5C,D), showing how the first carbohydrate plays an important role in recognition and the generation of binding affinity for the BMP-6–ACTR-I interaction The model also gives some hints as to why the binding affinity of BMP-6 for the type I receptors BMPRIA and BMPR-IB is not dependent on the presence of the carbohydrate structure The b1b2-loop of ActR-I is shortened by three residues in comparison to BMPRIA and BMPR-IB, possibly resulting in a less flexible loop in ActR-I Interactions between residues within this loop and the carbohydrate chain might thus contribute significantly to the binding free energy, whereas in the more flexible b1b2-loop of BMPR-IA ⁄ IB it does not In summary, our analysis shows the first structure of a BMP ligand member, which exhibits two vastly different conformations for the prehelix loop, which has been shown to be important for BMP type I receptor interaction Although, due to the lack of other BMP ligand–receptor complex structures with type I receptors A B C D Fig Model of the binary complex of N-glycosylated BMP-6 bound to ActR-I Ribbon representation of the binary complex of BMP-6 bound to its type I receptor ActR-I The carbohydrate chain [GlcNAcb1–4GlcNAcb1–4Man(a1,3Man)(a1,5Man)(b1,4Man)] – shown as thick lines – was added from a crystal structure analysis of BMP-7 (expressed in CHO cells) bound to the type II receptor ActR-II Several residues in the N-terminus, b1b2-loop or the loop in front of the a-helix of ActR-I are in close contact with the carbohydrate, namely the first two to three carbohydrate moieties, as would be predicted from our deglycosylation studies (A) Viewed from the top (B) Viewed from the side (C) BMP-6 (cyan ⁄ green) and ActR-I (magenta) are shown as surface representations to visualize the close packing of the carbohydrate in between the ligand–receptor interface; putative contact residues are indicated (D) Putative hydrogen bond interactions between ActR-I and the first GlcNAc residue of BMP-6 glycosylated at Asn73 Hydrogen bonds between the first carbohydrate residue (GlcNAc) and residues Lys11 and Tyr54 of ActR-I are shown 178 FEBS Journal 275 (2008) 172–183 ª 2007 The Authors Journal compilation ª 2007 FEBS S Saremba et al different from BMPR-IA, we cannot say whether the second noncanonical loop conformation plays a direct role in complex formation, however the second loop conformation confirms that this loop is highly mobile in BMP-6 and possibly also in other BMPs An NMR relaxation study on TGF-b3 has shown that this region is dynamically disordered in solution [33] As the binding epitope of BMPR-IA also seems to be not fully folded in solution, formation of complexes of BMP ligands and receptor seem to involve a large induced fit mechanism This conformational rearrangement upon binding might explain the promiscuous binding of BMPs, as it allows the binding epitopes to adapt to various different binding partners Furthermore, so far, functional analysis has highlighted the importance of the central hydrogen bond pair in BMP-2–BMPRIA for BMP type I receptor recognition [23] However, in the binding of BMP-6 to ActR-I, a new, so far unknown, main binding determinant has been discovered This new hot spot of binding involves an N-glycosylation motif conserved between BMP-2, BMP-7, and BMP-6, which is specifically required for binding of BMP-6 (and possibly BMP-7) to ActR-I but does not play a role in binding to the other type I receptors BMPR-IA and BMPR-IB This finding suggests that, in addition to the above-mentioned flexible binding epitope, usage of different main binding determinants might also add to the broad binding specificity observed in the BMP family Experimental procedures Expression and purification of recombinant proteins The mature part of hBMP-6, comprising amino acids 375– 513 plus an N-terminal extension MAPT (single-letter amino acid code) [34], was expressed in E coli Alternatively, a BMP-6 variant with residues 375–410 replaced with the sequence MAQAKHKQRKRLK was used (B2-BMP6) The protein was expressed in insoluble form in inclusion bodies BMP-6 isolated from these inclusion bodies was refolded and purified as previously described [35] Recombinant hBMP-6 obtained by eukaryotic expression, i.e CHO cells, was purchased from R&D Systems (Minneapolis, MN, USA) The extracellular domains of the receptors BMPR-IA and BMPR-IB were expressed as thioredoxinfusion proteins in E coli and purified as previously described [36] The extracellular domains of hActR-I (residues 21–123 [37]) and hActR-II (residues 18–135 [38]) were expressed in baculoviral-infected Sf9 insect cells as previously described [20] The receptor proteins hActR-I and hActR-II were purified by metal affinity chromatography Type I receptor specificity of BMP-6 using Ni–nitrilotriacetic acid–agarose (Qiagen, Hilden, Germany); the eluate was dialyzed against HBS buffer (10 mm Hepes, pH 7.4, 3.4 mm EDTA, 20 mm NaCl), and subjected to anion exchange chromatography The flowthrough of the latter step contains the monomeric, biologically active receptor protein, which was then finally purified by RP-HPLC Interaction analysis by surface plasmon resonance A BIAcore2000 system (BIAcore Life Science; GE Healthcare, Freiburg, Germany) was used for all biosensor experiments Ligand proteins were directly immobilized onto a CM5 biosensor chip at a density of about 800 resonance units (1 RU = pgỈmm)2), using the amine coupling kit (BIAcore Life Science; GE Healthcare) according to the manufacturer’s protocol Sensor chips were first activated by perfusing an ethyl-N-(3-diethylaminopropyl)carbodiimide (EDC) ⁄ N-hydroxysuccinimide (NHS) mixture for min; ligands were dissolved in 10 mm sodium acetate (pH 4.5) at a concentration of lgỈmL)1 and perfused over the activated chip surface until the required surface density was achieved Sensor chips were subsequently deactivated with m ethanolamine (pH 8.0) for All interaction experiments were carried out using HBS500 buffer (10 mm Hepes, pH 7.4, 500 mm NaCl, 3.4 mm EDTA, 0.005% surfactant P20) Sensorgrams of receptor–ligand interaction were recorded at a flow rate of 10 lLỈmin)1 at 25 °C The association and dissociation time was set to After each cycle, m MgCl2 was perfused for of regeneration Evaluation of recorded sensorgrams Apparent binding affinities were calculated using biaevaluation software 2.2.4 Bulk face effects, i.e unspecific binding to the biosensor or buffer exchange, were removed by subtracting a reference flow cell (FC1) from all sensorgrams Briefly, equilibrium binding constants of the interaction of BMP-2 with the type I receptors BMPR-IA and BMPR-IB were calculated by fitting the kinetic data to a : Langmuir binding model [KD(kin)], and those of the interaction of BMPs with hActR-I and hActR-II as well as of BMP-6 and BMP-7 with BMPR-IA and BMPR-IB were determined from the dose dependency of the equilibrium binding [KD(eq)] The relative standard deviations for mean KD(eq) values were below 25%; those of mean KD(kin) values were below 50% Deglycosylation of BMP-6 Recombinant hBMP-6 obtained from a eukaryotic expression system (R&D Systems, Minneapolis, MN, USA) was FEBS Journal 275 (2008) 172–183 ª 2007 The Authors Journal compilation ª 2007 FEBS 179 Type I receptor specificity of BMP-6 S Saremba et al fully or partially deglycosylated using either the endoglycosidase PNGase F (New England Biolabs, Frankfurt, Germany), or a mixture of the endoglycosidases PNGase F3 and PNGase H (New England Biolabs) For complete deglycosylation under nondenaturing conditions, U of PNGase F was used per microgram of BMP-6, with a reaction time of h at 37 °C For glycosylation trimming, a mixture of mU of PNGase F3 and mU of PNGase H was used per microgram of hBMP-6, with a reaction time of 24 h at 37 °C The completeness of the carbohydrate removal was analyzed by SDS ⁄ PAGE and MS Crystallization and structure analysis of BMP-6 Lyophilized E coli-derived wild-type BMP-6 and the variant B2-BMP-6 were dissolved in water at a concentration of 5–10 mgỈmL)1 and submitted to crystallization trials using Hampton Crystal Screens I and II (Hampton Research, Aliso Viejo, USA) Crystals were obtained using several sets of conditions and organic solvents, i.e 2-propanol, 2-methyl-2,4-pentanediol (MPD) or dioxane, or poly- ethylene glycols (polyethylene glycol 4000 to polyethylene glycol 6000) Wild-type BMP-6 and the B2-BMP-6 crystallized under identical conditions, however, due to the increased solubility of B2-BMP-6, reproducibly yielded larger crystals Suitable crystals of B2-BMP-6 grew from 25% MPD and 0.1 m sodium citrate (pH 4.0), and for wild-type BMP-6, the largest crystals were obtained from 20% 2-propanol and 0.1 m sodium citrate (pH 4.0) Diffraction data for B2-BMP-6 were collected from a single crystal at 100 K at the beamline XS06SA at the Swiss Light Source (SLS; Paul Scherrer Institute, Switzerland), and data for wild-type BMP-6 were acquired at 100 K using a home source (Rigaku RU300, MarResearch Imageplate 345, Osmic ConfocalBlue) Data were processed using xds software [39] or HKL2000 ⁄ Scalepack [40]; a summary of the processing statistics is given in Table Structure analysis was performed by applying molecular replacement using cns software [41] and the structure of BMP-7 (Protein Data Bank entry 1BMP [15]) as a search model The initial models were refined by iterative manual model building using quanta2006 software (Accelrys Inc., Table Processing and refinement statistics Statistical analyses for the highest-resolution shell are shown in parentheses Data processing B2-BMP-6 BMP-6 wild-typea Space group Unit cell P3121 ˚ a = b = 99.8 A, ˚ c = 86.8 A a = b = 90°, c = 120° 20.0–2.10 (2.25–2.10) 0.9183 229 655 (28 843) 26 690 (4397) 95.2 (95.7) 8.6 (6.6) 5.8 (28.2) 13.5 (4.1) P3121 ˚ a = b = 97.7 A, ˚ c = 85.1 A a = b = 90°, c = 120° 20.0–2.50 (2.59–2.50) 1.5418 62 200 (6058) 16 706 (1637) 99.4 (99.9) 3.7 (3.5) 5.1 (39.7) 13.4 (3.2) 20–2.10 (2.18–2.10) 25.9 (34.3) 27.9 (37.9) 20–2.50 (2.69–2.50) 23.5 (31.7) 27.0 (35.1) 0.007 1.475 0.883 64.2 0.32 75.1 0.007 1.153 0.844 49.8 0.42 73.8 85.8 (163) 13.2 (25) 1.1 (2) (0) 85.5 (159) 12.4 (23) 2.2 (4) (0) ˚ Resolution (A) ˚ Wavelength (A) Number of measured reflectionsb Number of unique reflectionsb Completeness (%) Multiplicity Rsym for all reflections (%) Refinement statistics ˚ Resolution (A) Rcryst (%) Rfree (%) (test set 5%) rmsd ˚ Bonds (A) Angles (°) Torsion angles (°) ˚ Average B-factor (A2) ˚ Cross-validated sigma coordinate error (A) Solvent content (%) Procheck analysisc Residues in most favored region (%) Residues in additional allowed region (%) Residues in generously allowed region (%) Residues in disallowed region (%) a Wild-type BMP-6 was analyzed for comparison The structures of BMP-6T2 and wild-type BMP-6 are identical within the accuracy of the resolution (rmsd of 0.7 for all Ca atoms); residues in the segment differing between BMP-6T2 and wild-type BMP-6 show no electron density, indicating a high degree of flexibility b Cut-off for reflections F > 0r c Number of residues is shown in parentheses 180 FEBS Journal 275 (2008) 172–183 ª 2007 The Authors Journal compilation ª 2007 FEBS S Saremba et al San Diego, CA, USA), and either Refmac5 [42] or CNS [41] was used for subsequent refinement Progress of refinement was monitored using the R-factors Rcryst and Rfree; the latter was calculated from a test dataset comprising 5% of randomly selected reflections In the final rounds of refinement, electron density difference maps Fobs – Fcalc were used to identify 78 water molecules (for wild-type BMP-6, 53 water molecules) and four MPD molecules (for wild-type BMP-6, 10 2-propanol molecules could be identified) The final minimization cycle yielded R-factors of 25.9 for Rcryst and 27.9 for Rfree for B2-BMP-6 (for wild-type BMP-6, Rcryst is 23.5 and Rfree is 27.0) ALP induction The teratocarcinoma AT508-derived cell line ATDC5 (RIKEN, Ibaraki, Japan, No RCB0565) was cultured in DMEM ⁄ F12 (1 : 1) medium containing 5% fetal bovine serum, and antibiotics (100 mL)1 penicillin G and 100 lgỈmL)1 streptomycin) For ALP assays, the cells were serum starved (2% fetal bovine serum) and exposed to ligands for 72 h in 96-well microplates After cell lyses, ALP activity was measured by p-nitrophenylphosphate conversion using an ELISA reader at 405 nm Type I receptor specificity of BMP-6 BMP-induced inhibition of INA6 cell proliferation Cells of the human myeloma cell line INA6 were seeded in DMEM in 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Kuszewski J, Type I receptor specificity of BMP-6 Nilges M, Pannu NS et al (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination Acta Crystallogr D Biol Crystallogr 54, 905–921 42 Murshudov GN, Vagin AA & Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method Acta Crystallogr D Biol Crystallogr 53, 240–255 FEBS Journal 275 (2008) 172–183 ª 2007 The Authors Journal compilation ª 2007 FEBS 183 ... ActR -I and ActR-II are faster (kon > 105ỈM)1Ỉs)1, koff > 10)1Ỉs)1), impeding the analysis of the dissociation rate and thus requiring analysis of the equilibrium binding ActR -I, is inactive in... interaction analysis yields the : interaction of BMPs and their receptor ectodomain proteins NB, no binding within detection limit (upper limit: KD > 400 lM) Ligands [affinity (lM)]a Receptor proteins... alterations of the type I receptor- binding profile were investigated Comparison of the receptor binding of Escherichia coli and CHO-cell derived BMP -6 reveals the importance of N-glycosylation for