Crystal structure analysis reveals a spring-loaded latch as molecular mechanism for GDF-5–type I receptor specificity
2009; Springer Nature; Volume: 28; Issue: 7 Linguagem: Inglês
10.1038/emboj.2009.37
ISSN1460-2075
AutoresAlexander Kotzsch, Joachim Nickel, Axel Seher, Walter Sebald, T. Müller,
Tópico(s)Connective tissue disorders research
ResumoArticle19 February 2009free access Crystal structure analysis reveals a spring-loaded latch as molecular mechanism for GDF-5–type I receptor specificity Alexander Kotzsch Alexander Kotzsch Lehrstuhl für Botanik I—Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany Lehrstuhl für Physiologische Chemie II, Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany Search for more papers by this author Joachim Nickel Joachim Nickel Lehrstuhl für Physiologische Chemie II, Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany Search for more papers by this author Axel Seher Axel Seher Lehrstuhl für Physiologische Chemie II, Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany Search for more papers by this author Walter Sebald Walter Sebald Lehrstuhl für Physiologische Chemie II, Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany Search for more papers by this author Thomas D Müller Corresponding Author Thomas D Müller Lehrstuhl für Botanik I—Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany Lehrstuhl für Physiologische Chemie II, Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany Search for more papers by this author Alexander Kotzsch Alexander Kotzsch Lehrstuhl für Botanik I—Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany Lehrstuhl für Physiologische Chemie II, Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany Search for more papers by this author Joachim Nickel Joachim Nickel Lehrstuhl für Physiologische Chemie II, Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany Search for more papers by this author Axel Seher Axel Seher Lehrstuhl für Physiologische Chemie II, Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany Search for more papers by this author Walter Sebald Walter Sebald Lehrstuhl für Physiologische Chemie II, Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany Search for more papers by this author Thomas D Müller Corresponding Author Thomas D Müller Lehrstuhl für Botanik I—Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany Lehrstuhl für Physiologische Chemie II, Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany Search for more papers by this author Author Information Alexander Kotzsch1,2,‡, Joachim Nickel2,‡, Axel Seher2, Walter Sebald2 and Thomas D Müller 1,2 1Lehrstuhl für Botanik I—Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany 2Lehrstuhl für Physiologische Chemie II, Theodor-Boveri-Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Würzburg, Germany ‡These authors contributed equally to this work *Corresponding author. Lehrstuhl für Botanik I—Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs Institut für Biowissenschaften (Biozentrum) der Universität Würzburg, Julius-von-Sachs Platz 2, 97082 Wuerzburg, Germany. Tel.: +49 931 888 6146; Fax: 49 931 888 6158; E-mail: [email protected] The EMBO Journal (2009)28:937-947https://doi.org/10.1038/emboj.2009.37 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Dysregulation of growth and differentiation factor 5 (GDF-5) signalling, a member of the TGF-β superfamily, is strongly linked to skeletal malformation. GDF-5-mediated signal transduction involves both BMP type I receptors, BMPR-IA and BMPR-IB. However, mutations in either GDF-5 or BMPR-IB lead to similar phenotypes, indicating that in chondrogenesis GDF-5 signalling seems to be exclusively mediated through BMPR-IB. Here, we present structural insights into the GDF-5:BMPR-IB complex revealing how binding specificity for BMPR-IB is generated on a molecular level. In BMPR-IB, a loop within the ligand-binding epitope functions similar to a latch allowing high-affinity binding of GDF-5. In BMPR-IA, this latch is in a closed conformation leading to steric repulsion. The new structural data now provide also a molecular basis of how phenotypically relevant missense mutations in GDF-5 might impair receptor binding and activation. Introduction Synovial joints are essential for the biomechanical function of the skeleton. As improper function, as observed in arthritic diseases, directly results in a severe loss of life quality, joint biology has been in focus of extensive research for years leading to an understanding of joint anatomy and histology as well as the biomechanical properties and roles of articular cartilage and other components in joint function and maintenance. However, little is known about how synovial joints acquire their structure in the developing embryo and in particular what factors are required for the differentiation of progenitor cells, which then give rise to each joint component (Pacifici et al, 2005). As a first sign of joint formation in the embryonic limb, an emergence of a mesenchymal interzone at each prospective joint site can be observed (Holder, 1977; Mitrovic, 1978). This interzone is a tripartite tissue structure composed of an intermediate cell layer and two outer cell layers with higher cell density. Interzone cells express a number of genes being involved in chondrogenesis such as Wnt-9a, Wnt-4, Noggin and growth and differentiation factor 5 (GDF-5) (Storm et al, 1994; Brunet et al, 1998; Hartmann and Tabin, 2001). GDF-5, a member of the large TGF-β superfamily of secreted growth factors, shows chondrogenic activity and congenital GDF-5 mutations cause defects in digit, wrist and ankle joints in mice and humans (Storm et al, 1994; Thomas et al, 1997). The expression of GDF-5 is most strikingly limited to regions where joints will develop and is one of the earliest markers of joint formation (Storm and Kingsley, 1999). Similar to other TGF-β superfamily members, GDF-5 binds to and oligomerizes two types of membrane bound serine-threonine kinase receptors termed type I and II. Upon ligand binding, these complexes transduce signals by phosphorylating members of the SMAD family of transcription factors, which upon activation enter the nucleus and regulate transcription of responsive genes (Massague, 1996). Recent experiments have implicated two different type I receptors in skeletal patterning, BMPR-IA and BMPR-IB. Both receptors are expressed in dynamic patterns during normal development. In several limb structures, for example, in joint interzones and perichondrium, an overlapping expression of BMPR-IA and BMPR-IB is observed (Mishina et al, 1995; Zou et al, 1997; Baur et al, 2000). With regard to the BMPR-IA and BMPR-IB expression patterns, GDF-5 signal transduction should be accomplished by the interaction with both BMPR-IA and BMPR-IB (Chang et al, 1994; Zou et al, 1997). Null mutations in the bmpr-1b gene produce viable mice with defects in bone and joint formation that closely resemble those seen in mice missing GDF-5 (Storm and Kingsley, 1996; Yi et al, 2000), whereas bmpr-ia−/− mice are known to die early in embryogenesis (Mishina et al, 1995). However, a conditional knockout of BMPR-IA under the control of a GDF5-Cre driver bypasses embryonic lethality and produces viable mice with normally formed joints. But, after birth articular cartilage within the joints wears away in a process reminiscent to osteoarthritis, which points at the importance of this receptor in cartilage homoeostasis and repair (Rountree et al, 2004). In the past, several single missense mutations in the mature part of the human GDF-5 have been described resulting in phenotypes such as brachydactyly A2 (BDA2), DuPan syndrome and symphalangism type I (SYM1) (for details, see Supplementary Table 6). Interestingly, the majority of phenotypically relevant mutations occur within a central loop (pre-helix loop) of the so-called wrist epitope of GDF-5 that represents the binding site for BMP type I receptors. The corresponding pre-helix loop in BMP-2 harbours the main binding determinants for type I receptor interaction. In contrast to BMP-2, which binds BMPR-IA and BMPR-IB with almost identical affinities (KD∼1–3 nM), GDF-5 binds BMPR-IB in vitro with about 10- to 20-fold higher affinity (KD∼1–2 nM) as compared with BMPR-IA (KD∼15–20 nM). A mutagenesis study revealed that a single residue in GDF-5 located in the pre-helix loop, Arg57, solely determines the binding specificity for the BMP type I receptor IB (Nickel et al, 2005). To date, neither the structure of GDF-5 bound to BMPR-IA nor to BMPR-IB has been reported. Only structure data for the unbound GDF-5 (Nickel et al, 2005; Schreuder et al, 2005) and other TGF-β members such as BMP-2 (Kirsch et al, 2000b; Keller et al, 2004; Allendorph et al, 2006; Weber et al, 2007), BMP-7 (Greenwald et al, 2003), activin-A (Thompson et al, 2003) or TGF-β3 (Hart et al, 2002; Groppe et al, 2008) in complex with either receptor subtype are currently available. A theoretical model for GDF-5 ligand–receptor complex based on these data has, however, failed to explain type I receptor specificity of GDF-5 (Nickel et al, 2005). Here, we present the crystal structure of GDF-5 bound to BMPR-IB allowing the deduction of mechanisms by which type I receptor specificity is encoded on a molecular level. Furthermore, these structural data allow for the first time an understanding of how phenotypically relevant missense mutations found in GDF-5 impair receptor binding and activation. Results Architecture of the complex of GDF-5 and its high-affinity type I receptor BMPR-IB The crystal structure of the binary complex GDF-5 bound to the extracellular domain of BMPR-IB was determined to 2.1 Å resolution. The final structure model was obtained by refining native data (Rmerge is 7.1% for all reflections and 35.4% for those in the highest resolution shell) and exhibits an Rfree of 25.6% and an Rcryst of 21.5% (for further processing and refinement statistics, see Table I). The asymmetric unit contains one GDF-5 monomer with one BMPR-IB ectodomain bound, thus the biological assembly is formed by a two-fold crystallographic symmetry axis resulting in a fully symmetrical dimer assembly. The GDF-5 dimer exhibits a butterfly-shaped architecture, with a central core formed by the cystine-knot flanked by the two α-helices from each monomer. Two β-sheets comprised of four strands each form the two fingers per monomeric GDF-5 subunit. Four receptor-binding sites exist in the GDF-5 dimer, the type II receptor sites (knuckle) are located at the back of the two fingers, whereas the type I receptor-binding sites (wrist) are located in the cleft between the helix α1 of one monomer and the front side of the fingers 1 and 2 of the other monomer. The two BMPR-IB molecules in the complex bind to the wrist epitopes of GDF-5 (Figure 1), resembling a similar ligand–receptor assembly as also found for BMP-2 when bound to BMPR-IA (Kirsch et al, 2000b; Keller et al, 2004). However, closer inspection reveals clear differences, with the BMPR-IB moved upward by almost 2 Å compared with the complex BMP-2:BMPR-IA (Figure 1). This coincides with a change in the tilt angle of about 9° when comparing a single receptor molecule (BMPR-IB versus BMPR-IA) in the binary ligand–receptor complexes of GDF-5 (this study) and BMP-2 (PDB entry 1REW) (Figure 1). The change becomes most apparent when the Cα atoms of both dimeric ligands GDF-5 and BMP-2 are superimposed (residues 12–69, 75–114 of BMP-2 and 17–74, 81–120 of GDF-5) yielding an r.m.s.d. of only 0.86 Å, indicating that the structures of the ligands are highly similar. In contrast, the two receptor ectodomains of BMPR-IA and BMPR-IB are clearly differently placed in the wrist epitope of both superimposed complexes. A line through the Cα atoms of Phe66 (Phe85 in BMPR-IA), which marks the centre of rotation and Cys82 of BMPR-IB and the equivalent Cys101 in BMPR-IA in the central β-sheet shows that a single BMPR-IB rotates upward by an angle of 9°. Although this change in location and orientation for BMPR-IB in the wrist epitope of GDF-5 (compared with BMP-2:BMPR-IA) is far less pronounced compared with the differences found for the TGF-β type I receptor in the TGF-β3:TβR-II:TβR-I complex (Groppe et al, 2008), it clearly shows that the location and orientation of the receptor ectodomains in the ligand-binding sites of different BMPs can vary. Figure 1.Architecture of the complex of GDF-5 bound to BMPR-IB. (A) Ribbon representation of the full tetrameric complex of GDF-5 dimer (in blue and green) bound to the extracellular domains of two BMPR-IB molecules (red). A stippled line indicates the crystallographic two-fold axis. (B) As in (A), but viewed from the top. (C) The complex structures of GDF-5:BMPR-IB and BMP-2:BMPR-IA (PDB 1REW) were structurally aligned using the Cα atoms of both ligand dimers and the program Quanta2006. The ligand dimer superposition exhibits an r.m.s.d. of 0.86 Å (Cα of GDF-5: 17–74, 80–120 versus BMP-2: 12–74, 75–114). The ligand superposition clearly reveals that BMPR-IA and BMPR-IB are shifted in both complexes up to 2 Å. Further inspection shows that the BMPR-IB molecule (red) is tilted by 9° (angle between Cys82(BMPR-IB)–Phe66(BMPR-IB)–Cys101(BMPR-IA), see line) towards finger 2 of GDF-5 compared with the orientation of BMPR-IA (magenta) in complex with BMP-2. Residue Phe66 (Phe85 in BMPR-IA) presents the centre of rotation. (D) Despite the reorientation the core structures of both type I receptors are identical (r.m.s.d. 0.7 Å for β-sheet core without β1β2, β3β4 and α1β5 loops), only the β1β2 and α1β5 loops differ significantly. Download figure Download PowerPoint Table 1. Data collection and refinement statistics for the GDF-5:BMPR-IB complex structure MAD dataa Native dataa Processing Space group P42212 P42212 Unit cell a=b=76.62, c=82.12 a=b=76.46, c=82.78 α=β=γ=90° α=β=γ=90° Wavelength (Å) 0.97979 (inflection) 0.97962 (peak) 0.90789 (remote) 1.10485 (native) Resolution (Å) 36.32–2.90 (3.00–2.90) 38.31–2.60 (2.69–2.60) 36.26–2.90 (3.00–2.90) 34.19–2.10 (2.18–2.10) Rmerge 15.7 (49.2) 10.0 (40.8) 12.0 (39.0) 7.1 (35.4) I/σI 6.2 (2.5) 9.1 (3.5) 8.6 (3.7) 14.7 (5.3) Completeness (%) 99.8 (100.0) 99.9 (100.0) 99.9 (100.0) 99.5 (100) Redundancy 6.69 (6.93) 6.75 (6.85) 6.1 (6.3) 9.7 (9.9) Resolution (Å) 34.19–2.10 No. of reflections 14 062 Rcryst/Rfree (%) 21.5 (28.9)/25.6 (24.2) Protein 1484 Water 52 Protein (Å2) 71.2 Water (Å2) 60.9 Bond length (Å) 0.014 Bond angles (deg) 1.340 Values in parentheses are for the highest resolution shell. a One crystal was used to collect the diffraction data. The structure of the binding loop of BMPR-IB differs from that of BMPR-IA Our complex structure GDF-5:BMPR-IB now yields data for a BMP type I receptor ectodomain other than BMPR-IA and thus allows to detect structural differences and variability among BMP type I receptors. The ectodomain of BMPR-IB shares about 50% identity on amino-acid sequence level with BMPR-IA (Supplementary Figure 1). Therefore, many of the secondary structure elements and the tertiary fold are conserved between BMPR-IB and BMPR-IA (Figure 1). However, a detailed comparison of the structures of both receptor ectodomains reveals some structural differences between BMPR-IB and BMPR-IA (PDB entry 1REW). The structural core comprising five β-strands superimposes well showing an r.m.s.d. of 0.7 Å, but considering all Cα in the ectodomains the r.m.s.d. rises to 2.2 Å, showing that the loop sections differ significantly between BMPR-IB and BMPR-IA. As binding and recognition by GDF-5 are mainly mediated through the β1β2 and the α1β5 loops of BMPR-IB this is of important biological consequence. Taken together, both loops contribute almost 80% of the buried surface area of the BMPR-IB ectodomain in the complex. These loops show large structural differences between BMPR-IB and BMPR-IA with Cα positions being shifted up to 3 Å in the β1β2 loop and up to 5 Å in the α1β5 loop. Thus, the binding determinants of the GDF-5:BMPR-IB and BMP-2:BMPR-IA (PDB entry 1REW) complexes likely differ not only due to the different orientations of the type I receptor ectodomains but also due to the differences present in the binding loops of BMPR-IB and BMPR-IA. Notably, the length of the α-helix, which carries the hot spot of binding for the BMP-2:BMPR-IA interaction, also varies between BMPR-IB and BMPR-IA. In BMPR-IA, the α-helix comprises residues Ser83 to Lys88 and hence has a length of more than 1.6 turns. In BMPR-IB, the α-helix is two residues shorter (Ser64 to Gln67) and consists of just one turn. The GDF-5–type I receptor interaction in the GDF-5:BMPR-IB complex About 1040 Å2 solvent accessible surface of the BMP type IB receptor (for one receptor ectodomain of the dimeric assembly) and 1100 Å2 solvent accessible surface of GDF-5 (per type I receptor-binding site) are buried upon complex formation. Thus, considering the dimeric assembly with two BMPR-IB and two GDF-5 wrist epitopes involved, about 4280 Å2 of the protein surfaces are buried upon binding of GDF-5 to two BMPR-IB. Here, 22 residues of BMPR-IB (per molecule) and 21 residues of GDF-5 (per monomeric subunit) mark the contact interface. Of the 22 contact residues of BMPR-IB and the 21 contact residues of GDF-5 in the GDF-5:BMPR-IB interface, 15 are conserved with BMPR-IA and 13 are conserved with BMP-2 in the BMP-2:BMPR-IA interface (Figure 2). Nine intermolecular hydrogen bonds (H-bonds) are observed in the GDF-5:BMPR-IB contact (Supplementary Table II). Five of these involve residues in helix α1 and the α1β5 loop of BMPR-IB, suggesting that these elements are highly important for ligand recognition and binding. Interestingly, the bi-dentate H-bond between the conserved glutamine in helix α1 of the type I receptor (BMPR-IB:Gln67; BMPR-IA:Gln86) and the main chain polar groups of a conserved leucine in the pre-helix loop of the ligand (GDF-5 Leu56; BMP-2 Leu51) is also present in the GDF-5:BMPR-IB contact. However, whether this H-bond, similar as for the BMP-2:BMPR-IA interaction (Keller et al, 2004), presents the hot spot of binding for the GDF-5:BMPR-IB complex cannot be told from the structure. Figure 2.Interface of the GDF-5:BMPR-IB complex. (A) Ligplot (Wallace et al, 1995) analysis of the GDF-5:BMPR-IB interface. H-bonds are indicated as stippled lines with the residues involved shown as ball-and-stick models. Hydrophobic contacts are presented as spheres; the buried surface area of each contact residue is given in Å2 (boxed values below residues). Only hydrophobic contacts with buried surface areas ⩾5 Å2 are shown. (B) 'Open book' view of the GDF-5:BMPR-IB complex with BMPR-IB rotated out of the interface by a 120° rotation in the y axis. The surface is colour coded by amino-acid polarity; red marks negatively charged residues, blue positively charged residues, green indicates polar uncharged amino acids and grey colour represents hydrophobic amino acids. Contact residues are indicated by residue number and amino-acid type in one-letter code. The contact surface of BMPR-IA in the BMP-2:BMPR-IA complex (PDB entry 1REW) is given for comparison. Download figure Download PowerPoint Therefore, Gln67 and Phe66 in BMPR-IB were mutated to alanine, and their binding to GDF-5 and BMP-2, was tested by SPR. Surprisingly, the surmised hot spot of binding Gln67 showed only slightly decreased affinities for GDF-5 (6.3-fold) and BMP-2 (4.5-fold), respectively (Table II). Thus, in contrast to BMPR-IA, where the mutation BMPR-IAQ86A leads to almost 100-fold loss in affinity for BMP-2 (Keller et al, 2004), the conserved glutamine does not represent a hot spot of binding for BMPR-IB. Exchange of Phe66 in BMPR-IB by alanine, however, almost completely abolished binding to GDF-5 and BMP-2. Hatta et al (2000) observed that the affinity of the BMPR-IA variant F85A is decreased for BMP-2 only 15-fold (ΔΔG=1.5 kcal mol−1), suggesting that Phe85 is not a hot spot of binding in the BMP-2:BMPR-IA interaction. Thus, the conserved phenylalanine is crucial only for binding of BMPR-IB to BMPs, whereas the conserved glutamine seems important only for binding of BMPR-IA to the ligands. These findings corroborate our hypothesis that recognition and binding of BMPR-IB to BMPs differ from BMPR-IA. Table 2. Binding affinities of GDF-5 and BMP-2 to immobilized BMPR-IB variants GDF-5 BMP-2 KD (nM)a ΔΔG (kcal mol−1)b KD (nM)a ΔΔG (kcal mol−1)b BMPR-IB 1.3±0.55 — 4.8±1.80 — F66A ⩾1000c ⩾4.0 n.b.d ⩾4.5 Q67A 8.2±2.98 (6.3 × ) 1.1 21.7±11.31 (4.5 × ) 0.9 H22S/H23G 4.1±1.88 (3.2 × ) 0.7 7.2±1.21 (1.5 × ) 0.2 a The apparent binding constant KD was derived from calculating KD=koff/kon. Numbers in parentheses represent the relative change compared with wild-type BMPR-IB. b Calculated using ΔΔG=(−RTlnKD)wt−(−RTlnKD)var with R=1.98 cal mol−1 K−1 and T=293.15 K. Values ⩾2.0 kcal mol−1 identify a hot spot of binding. c The apparent KD was estimated from the dose dependency of equilibrium binding and presents the lower limit due to technical limitations of the BIAcore2000 system. d No binding above background levels could be detected, from the highest analyte concentration applicable in the analysis, the binding affinity was estimated to be ⩾10 μM. GDF-5 passes through an induced fit upon complex formation Comparison of free GDF-5 and GDF-5 bound to BMPR-IB shows that GDF-5 passes through an induced fit upon complex formation. Both fingers of GDF-5 move towards BMPR-IB upon binding, indicating that the opening of the wrist epitope is wider in the free form and upon BMPR-IB binding the fingers 'wrap around' BMPR-IB to make packing tighter (Figure 3). Figure 3.Structural rearrangements in GDF-5 upon complex formation. (A) Superposition of free (grey, PDB entry 1WAQ) and GDF-5 bound to BMPR-IB (green) showing the structural rearrangements upon receptor binding. Regions of interest are highlighted in b, c and d. Distances between Cα positions are indicated. (B) BMPR-IB binding leads to shifts of up to 4 Å in fingers 1 and 2 of GDF-5. (C) The tryptophans 33 and 36 of GDF-5 change their side chain conformation upon type I receptor binding. (D) The C-terminal end of the α-helix moves towards the β-sheet of GDF-5 by 1.5 Å; also the pre-helix loop undergoes an induced fit upon BMPR-IB binding. Download figure Download PowerPoint The finger 2 of GDF-5 moves as a rigid body with the side chains being pre-oriented in the free form. In contrast, in finger 1 of GDF-5 several side chains move significantly. In unbound GDF-5, Trp36 is pointing away from the receptor-binding epitope. In the complex, the Trp36 points towards the receptor, with the side chain atoms moving by almost 8 Å (Figure 3). These large rearrangements in the ligand are not observed in the BMP-2:BMPR-IA complex formation. In BMP-2, the movements in fingers 1 and 2 are less than 2 Å with the side chains being pre-oriented before receptor binding. The GDF-5 pre-helix loop also rearranges upon BMPR-IB binding. The changes in Cα position are comparable to those in BMP-2 upon binding to BMPR-IA (free BMP-2: 3BMP; bound BMP-2: 1REW). On the basis of its homology to BMPR-IA, we also think that BMPR-IB passes through an induced fit mechanism upon ligand binding. NMR studies on the extracellular domain of free BMPR-IA showed that the β1β2- and especially the β4β5- loops are highly flexible and disordered in solution (Klages et al, 2008). Helix α1 is absent in free BMPR-IA (PDB entry 2K3G). As the helix of bound BMPR-IB is even shorter compared with BMPR-IA and thus probably less stable, this loop segment is likely similarly flexible in free BMPR-IB. Thus, both complex partners bind by an induced fit mechanism, which possibly represents a molecular mechanism to generate specificity as well as promiscuity. The BMPR-IB specificity of GDF-5 is based on a spring-loaded latch in BMPR-IB In contrast to BMP-2, GDF-5 is described to have a pronounced type I receptor specificity in vivo (Nishitoh et al, 1996). In vitro, type I receptor discrimination of GDF-5 seems less dramatic with respect to affinities for BMPR-IB (KD 1.3 nM) and BMPR-IA (KD 16.2 nM), showing that only a factor of 10–20 is sufficient for discrimination between BMPR-IB and BMPR-IA (Nickel et al, 2005). However, even this supposedly small difference is of great physiological significance for GDF-5 function in vivo as can be seen from the R57L mutation (R438L pro-protein numbering). The mutant GDF-5R57L exhibits an enhanced affinity for BMPR-IA (KD 4.2 nM) and nearly unaltered binding to BMPR-IB (KD 0.7 nM) (Table III). This correlates with the mutation of Arg57 to alanine that abrogates type I receptor specificity of GDF-5 completely (Nickel et al, 2005). Therefore, the mechanism encoding for BMPR-IB specificity should be in close proximity of GDF-5 Arg57. In the complex structure, GDF-5 Arg57 is completely hidden inside the ligand–receptor interface and shares contact with several residues of BMPR-IB. This is in contrast to the modelling studies, that suggested Arg57 pointing away from the interface out into the solvent (Nickel et al, 2005). Table 3. Binding affinities (KD (nM)) of GDF-5 variants to immobilized BMPR-IAec, -IBec and -IIec BMPR-IAa BMPR-IBa BMPR-IIb GDF-5 16.2±6.38 1.3±0.62 65.8±4.2 GDF-5 R57L 4.2±1.51 0.7±0.16 55.9±3.8 GDF-5 R57A 2.0±0.68 0.6±0.19 72.4±8.4 GDF-5 L60P n.b.c 42.3±7.32 31.9±5.9 GDF-5 ΔL56+S58T+H59L ⩾1000d ⩾1000d 73.0±5.4 a The apparent binding constant KD was derived from calculating KD=koff/kon. b KD values were evaluated from the dose dependency of equilibrium binding. c No binding above background levels could be detected. d The values for the apparent KD represent the lower limit estimated from the highest analyte concentration used in the SPR analysis. During refinement, two alternative conformations were detected for the β1β2 loop of BMPR-IB. As there is only one receptor ectodomain in the asymmetric unit—the full tetrameric GDF-5:(BMPR-IB)2 complex is formed by a two-fold crystallographic axis—both conformations are observed in the same BMPR-IB molecule (Figure 4). In the 'closed' conformer, the β1β2 loop of BMPR-IB contacts Arg57, with the Arg side chain being clamped between the aromatic ring of Phe41 and the backbone of His23 and His24 of BMPR-IB. In this conformer, Arg57 is shielded from solvent and forms two H-bonds with the backbone carbonyl of His23 and His24 of BMPR-IB (see also Supplementary Figure 2). The second conformer presents an 'open' conformation, in which the backbone of this section of the β1β2 loop moves away from Arg57 towards the solvent. This movement is due to a 180° flip in the Psi torsion angle of His22 resulting in an altered backbone route for BMPR-IB His23 and His24 (Supplementary Figure 3). The Cα atoms of the latter histidines relocate by 2.3 Å, possibly allowing water to fill the cleft nearby GDF-5 Arg57. The side chain conformation of Arg57 is unchanged in both conformations. As at the resolution of 2.1 Å, it is not reasonable to do occupancy refinement, the relative populations for both conformers can be estimated only from the electron density, suggesting that both conformations are roughly equally populated. Figure 4.A spring-loaded latch in BMPR-IB for high-affinity binding to GDF-5. (A) Stereoview of the two alternative conformations of the BMPR-IB β1β2 loop in the GDF-5:BMPR-IB complex. The electron density is contoured at 0.8σ. The open conformation is shown with the C atoms coloured in green and in the closed conformation the C atoms are shown in cyan. Arg18 of GDF-5 (grey), which also adopts two alternative conformations, is labelled in red. (B) Scheme (stereoview) of the two alternative β1β2 loop conformations (colour code for BMPR-IB as in (A), GDF-5 is in pale green) and their interaction with the specificity-determining residue of GDF-5 Arg57. In the closed conformation, two H-bonds are formed between the β1β2 loop and Arg57 of GDF-5. (C) Docking of BMPR-IA (magenta C atoms) onto BMPR-IB reveals that the BMPR-IA β1β2 loop will cause a steric clash with GDF-5 Arg57, if same conformation for the BMPR-IA β1β2 loop as in the complex BMP-2:BMPR-IA is assumed. Download figure Download PowerPoint The local conformational change in the β1β2 loop possibly results from fixing the segment His22 to His24 in between the disulphide bond Cys21–C
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