Crystal structure of human bone morphogenetic protein-2 at 2.7 Å resolution1

Article No. jmbi.1999.2590 available online at http://www.idealibrary.com on

J. Mol. Biol. (1999) 287, 103±115

Crystal Structure of Human Bone Morphogenetic Ê Resolution Protein-2 at 2.7 A Clemens Scheufler, Walter Sebald* and Martin HuÈlsmeyer University of WuÈrzburg Physiological Chemistry II 97074 WuÈrzburg, Germany

Homodimeric bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor b (TGF-b) superfamily that induces bone formation and regeneration, and determines important steps during early stages of embryonic development in vertebrates and non-vertebrates. BMP-2 can interact with two types of receptor chains, as well as with proteins of the extracellular matrix and several regulatory proteins. We report here the crystal structure of human BMP-2 determined by molecuÊ resolution. lar replacement and re®ned to an R-value of 24.2 % at 2.7 A A common scaffold of BMP-2, BMP-7 and the TGF-bs, i.e. the cystineknot motif and two ®nger-like double-stranded b-sheets, can be superimÊ . In contrast to the TGF-bs, posed with r.m.s. deviations of around 1 A the structure of BMP-2 shows differences in the ¯exibility of the N terminus and the orientation of the central a-helix as well as two external loops at the ®ngertips with respect to the scaffold. This is also known from the BMP-7 model. Small secondary structure elements in the loop regions of BMP-2 and BMP-7 seem to be speci®c for the respective BMPsubgroup. Two identical helix-®nger clefts and two distinct cavities located around the central 2-fold axis of the dimer show characteristic shapes, polarity and surface charges. The possible function of these speci®c features in the interaction of BMP-2 with its binding partners is discussed. # 1999 Academic Press

*Corresponding author

Keywords: BMP-2; X-ray crystallography; bone morphogenetic protein; TGF-b family; cystine-knot

Introduction Bone morphogenic protein-2 (BMP-2) is one of the main representatives of a group of bone morphogenetic proteins that were originally de®ned by their bone and cartilage-inductive activity at nonskeletal sites in vivo (Urist, 1965; Sampath & Reddi, 1981). Cloning of the cDNA (Wozney et al., 1988) revealed that BMP-2, like other members of the family (Celeste et al., 1990; Ozkaynak et al., 1990), belongs to the transforming growth factor b (TGFb) superfamily. The TGF-b superfamily is a group of multifunctional cytokines that play important Present address: C. Scheu¯er, Max-Planck-Institute for Biochemistry, 82151 Martinsried, Germany. Abbreviations used: BMP-2, bone morphogenic protein-2; TGF-b, transforming growth factor b; MPD, 2-methylpentane-2,4-diol; VEGF, vascular endothelial growth factor; NGF, nerve growth factor; BNDF, brainderived neurotrophic factor. E-mail address of the corresponding author: [email protected] 0022-2836/99/110103±13 $30.00/0

roles in development and in the control of differentiation and proliferation. Accordingly, BMP-2 is synthesized as a 453 residue proprotein, which becomes glycosylated, proteolytically cleaved and dimerized to yield the mature homodimeric protein consisting of the 114 C-terminal proprotein residues. Like all members of the TGF-b superfamily, BMP-2 contains the conserved cystine-knot folding motif (McDonald & Hendrickson, 1993; Murray-Rust et al., 1993). The sequence preceding the ®rst cystine (pro-knot sequence) of BMP-2 has been shown to be involved in heparin binding (Koenig et al., 1994; Ruppert et al., 1996). In contrast to the TGF-b factors, this sequence is not covalently attached to the protein core by a disul®de bridge. Excluding the pro-knot residues, the amino acid sequence of BMP-2 shows 92 % identity with that of BMP-4. The BMP-2/4 subgroup is highly conserved. Homologs have been identi®ed from Drosophila (dpp; Newfeld et al., 1997), Xenopus (xBMP-2/4; Plessow et al., 1991), zebra®sh (zBMP2/4; Martinez-Barbera et al., 1997) and ascidia (HrBMPa/b; Miya et al., 1997). For these proteins, # 1999 Academic Press

104 decisive roles during multiple steps in animal development, like dorsoventral pattern formation, organ and tissue development and limb/wing formation have been described (reviewed by Hogan, 1996). BMP-2 transmits signals into the cell by binding and oligomerizing two types of receptor chains (Reddi, 1997). Possibly, two type 1 and two type 2 chains interact with one BMP-2 molecule during receptor activation, as in the TGF-b receptor system (Wrana et al., 1994). Cross-linking experiments employing iodinated BMP-2 or BMP-4 revealed interactions with several different type 1 receptors, as ALK-3 (BMPR-IA), ALK-6 (BMPR-IB) and ALK-2 (ActR-IA), and with type 2 receptor chains BMPR-II and ActR-II (Liu et al., 1995; ten Dijke et al., 1994b). The latter receptors, however, reacted to a larger extent only when the type 1 receptor was present (Yamashita et al., 1995). The speci®cities and af®nities reported for the interactions between BMP-2/4 and type 1 receptors are still under discussion (Chalaux et al., 1998), in particular because the cross-linking ef®ciency may not necessarily re¯ect the functional binding of the ligand (ten Dijke et al., 1994a). Interestingly, binding of BMP-2/4 in the 100 pM range has been found for the type 2 receptor daf4 from Caenorhabditis elegans (Estevez et al., 1993), and for the extracellular domain of the TGF-b type 2 receptor (Goetschy et al., 1996). The BMP-2/4 subgroup shows about 50 % sequence identity with related BMP-5/6/7 (OP-1)/8 (OP-2), that have been also called the A60 subgroup due to the similarity with the Drosophila A60 protein (Kingsley, 1994). Mammalian BMP-7 determines the development of several organs (kidney, eye). The in vivo function is clearly different from that of BMP-2, as seen by the effect of gene deletion studies in mice (Dudley et al., 1995; Luo et al., 1995). The de®ciency of BMP-2 results in embryonic lethality between day 7.5 and 9.5 of gestation (Zhang & Bradley, 1996), whereas the inactivation of the BMP-7 gene leads to death at birth due to renal failure. BMP-7 binds to the ALK-2 (ActR-IA) and ALK-6 receptor chains (Liu et al., 1995; ten Dijke et al., 1994b) as well as to BMPR-II and ActR-II (Yamashita et al., 1995) in the presence of a type 1 receptor. The crystal structure of BMP-7 (OP-1) resolved Ê (Grif®th et al., 1996) revealed that the overat 2.8 A all structure of a member of the BMP family is similar to that of TGF-b2 (Daopin et al., 1992; Schlunegger & Grutter, 1992) or TGF-b3 (Mittl et al., 1996). A dimeric scaffold provided by two cystine knots and b-strands of both monomers are virtually superimposable, despite the fact that the proteins of the two families share only 30 to 35 % sequence identity. Differences became apparent in the folding of the N-terminal pro-knot sequence as well as in the orientation of two loop regions and a central a-helix. In addition, speci®c and distinct patterns of surface charge and hydrophobicity have been observed. The present crystal structure

Crystal Structure of BMP-2

of human BMP-2 extends three-dimensional structure information to a member of another subgroup of the BMP family that has been extensively studied in embryonal development as well as in bone repair and bone induction. The three-dimensional model of BMP-2 will be useful in further functional analysis of the interaction of BMP-2 with receptor chains, glycosaminoglycans, and with regulatory proteins such as chordin and noggin (Reddi et al., 1997).

Results and Discussion Quality assessment Ê solvent ¯attened and weighted The ®nal 2.7 A electron density map allowed the location of 838 protein atoms (104 residues), 33 water molecules and one molecule of 2-methylpentane-2,4-diol (MPD). The surface loop (residues 48-55) located before helix a3 is weakly de®ned in the 2Fo ÿ Fc electron density map. The N terminus (residues 1-8) has no interpretable electron density probably due to disorder. The ®rst eight residues were therefore excluded from the model. Four sidechains had weak electron density: Arg9, Leu10, Lys11, Glu46. All weakly de®ned residues are located at the protein surface and show expected ¯exibility. With the exception of Phe41, a detailed analysis of f/c-angles assigns all other nonglycine residues in the ``most favored'' or ``additional allowed regions'' (Laskowski et al., 1992) of the Ramachandran plot (Ramachandran & Sasisekharan, 1968). The R-value for the model is 24.2 % (Rfree 27.8 %) Ê resolution. The average coordinate error as at 2.7 A estimated from a cross-validated sA-plot (Brunger, Ê . The average B1997; Brunger et al., 1998) is 0.55 A Ê 2. This value is relafactor for all atoms is 35.2 A Ê2 tively high compared to values of around 20 A usually found in globular proteins, but it is not Ê 2; unusual in the TGF-b superfamily: TGF-b2 33.1 A 2 2 Ê Ê TGF-b3 29.6 A ; BMP-7 28.5 A . The elevated B-factors might re¯ect an unusual higher molecular mobility in the TGF-b family due to a missing rigid protein core which is typical for globular proteins (see below). Another cause for this result may result from the loose crystal packing as shown by a Ê 3/Da. Matthews parameter (VM) of 3.0 A A summary of the re®nement statistics is given in Table 1. Overall description and monomer fold In its biologically active form BMP-2 is a homodimer. Each monomer is a single-domain protein and consists of 114 residues. The three-dimensional structure and especially the symmetry of the dimeric protein resembles a section mark. The Ê  35 A Ê  30 A Ê. dimensions of the dimer are 70 A Ê The middle part of the monomer is only 10 A thick, corresponding to only one layer of a b-sheet.

105

Crystal Structure of BMP-2 Table 1. Data processing and re®nement statistics of BMP-2 A. Crystals and data processinga Space group Unit cell Ê) Resolution (A Number of collected reflexions Number of unique reflexions Completeness (%) Multiplicity Intensities (I/s)

R32 Ê , c ˆ 107.75 A Ê , a ˆ b ˆ 90 , g ˆ 120 a ˆ b ˆ 91.44 A 20-2.7 (2.8-2.7) 12,853 (873) 4645 (407) 94.6 (84.4) 3.0 (2.2) 24.2 (5.4)

Rmerge (%)

4.1

(12.3)

B. Refinement statistics Rcryst (%) Rfree (%) Ê) r.m.s. bond length (A r.m.s. bond angle (deg.) r.m.s. dihedrals (deg.) r.m.s. impropers (deg.) Ê 2) Average B-value (A Ê) Coordinate error (cross-validated sigma) (A a b

24.2 27.8 0.01 1.30 23.5 1.00 35.2 0.55

Values as de®ned in XDS (Kabsch, 1993), values in parentheses represent data from the highest resolution shell. Values as de®ned in CNS (Brunger et al., 1998).

Each of the BMP-2 monomers contains a cystineknot which is built by six cysteine residues forming three intrachain disul®de bridges. Cys43, Cys47, Cys111 and Cys113 participate in an eight-membered macrocycle wide enough for the last cystine bridge to pass through (Cys14/Cys79, Figure 1). Similar cystine-knots have been found in transforming growth factors (TGF-b1, TGF-b2, TGF-b3), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), b nerve growth factor (bNGF), brain-derived neurotrophic factor (BNDF) and gonadotropin. These proteins are members of the cystine-knot growth factor super-

family (McDonald & Hendrickson, 1993; MurrayRust et al., 1993). The cystine-knot is a structurally highly conserved feature as seen from r.m.s. deviÊ , which is ations typically in the range of 0.5 A within the magnitude of error (data not shown). As the proteins of this family lack the common hydrophobic core of globular proteins, the rigid cystine-knot scaffold is necessary to stabilize the entire structure. Further stabilization of the BMP-2 structure is achieved by dimerization which creates a hydrophobic core between the monomers (see below).

Figure 1. Stereoview of the BMP-2 cystine-knot. The displayed 2mFo ÿ DFc electron density map has been contoured at 1s. All main-chain and side-chain atoms are clearly de®ned by density. The cystine bridges between Cys34/Cys111 and Cys47/Cys113 form a ring-like structure with the main chain. The ring is wide enough for a third disul®de bridge to pass through (Cys14/Cys79). Cys78, visible in the top part of the Figure, forms the interchain cystine bridge to the neighboring subunit.

106 The TGF-b-like proteins can be assigned to several subgroups (Grif®th et al., 1996), but to date only few of these proteins have been characterized by structure models: in the TGF-b subgroup these are TGF-b1 (NMR study, Hinck et al., 1996), TGF-b2 (Daopin et al., 1992; Schlunegger & Grutter, 1993) and TGF-b3 (Mittl et al., 1996); in the BMP/OP-subgroup, so far only BMP-7 has been studied (Grif®th et al., 1996). BMP-2 is the ®rst member of the BMP-2/4 subgroup structurally characterized to date. As expected, it shares the folding topology of the TGF-b superfamily (Figure 2). The typical features of these proteins are two separated antiparallel b-sheets, the second of which adopts a twisted crossover conformation, and a four-turn a-helix approximately perpendicular to the strands. The b-sheets can be further divided into nine b-strands (Figure 3(a)), but they do not form a continuous four-stranded sheet because they are too far apart for hydrogen bonding. The overall folding topology has been described as a hand (Daopin et al., 1992) with the helix mimicking the wrist, the cystine-knot core the palm, and the b-sheets the ®ngers. There are clear differences in some structural elements between the TGF-bs and the BMPs (Figure 3(a)): the N terminus is not visible in the electron density either in BMP-2 or in BMP-7 due to disorder. In contrast, TGF-b2 and TGF-b3 exhibit a short N-terminal a-helix (a1), that is anchored to the protein core by an additional cystine bridge. Moreover, BMP-2 and BMP-7 do not contain the short helix a2 that can be observed after the second b-strand in the TGF-bs. This is due to a one amino acid deletion in the BMPs (Figure 3(a)) which aligns with Arg26 (a2) in TGF-b2 and results

Crystal Structure of BMP-2

in a tighter non-a-helical turn. As a consequence, conserved tryptophan residues (Trp28 and Trp31 in the case of BMP-2) adopt a similar position in all proteins. This deletion seems to be a conserved feature among known BMPs, GDFs and inhibins (Kingsley, 1994). The structure model presented here reveals two elements existing only either in BMP-2 or BMP-7, and may therefore represent characteristic features of the respective BMP-subgroup: BMP-2 forms a short b-strand (b5a) in the loop before the long helix a3. This might be the result of a three residue insertion before helix a3 compared to the TGF-bs, forcing the loop to adopt a wider conformation. Although BMP-7 has the same number of amino acid residues in this loop before helix a3, comparable b-strand conformations are missing. On the other hand, helix a4 in ®ngertip 2 is uniquely found in BMP-7. The ®ngertips are built by loops of different length: loop 1 connecting b2 and b4 is a long

-loop, which contains 14 residues in all TGF-b proteins (Phe22-Pro35 in BMP-2). Loop 2 connecting b7 and b8 can be described as a relatively narrow hairpin loop with 2:2 conformation (Sibanda & Thornton, 1991) in the case of BMP-2 and BMP-7, and 3:5 conformation in the case of TGF-b2 and TGF-b3. Phe41 is a key residue in the BMP-2 structure model (Figure 2) . This residue is the only one that adopts unusual f/c-angles (f ˆ 67.2  , c ˆ 177.8  ) in the Ramachandran plot (Ramachandran & Sasisekharan, 1968), but it is clearly de®ned by electron density. Phe41 is the starting point of strand b5 (Figure 3(a)), forming a main-chain hydrogen bond with Leu19 in b2 (Phe41Ê ). This is the only region in the N


Leu19-O: 3.1 A

Figure 2. Stereoview of the folding topology of the native BMP-2 dimer: a-helices are indicated as spiral, b-strands as arrow, disul®de bridges are shown as green sticks. The subunits are color-coded blue and orange, respectively. The interactions that are responsible for dimer formation between helix a3 and the b-strands of the other subunit are clearly visible. cis-Pro35 and Phe41 are indicated in dark gray. The unique b-strand b5a is located near helix a3. The Figure was produced with Ribbons (Carson & Bugg, 1986).

Crystal Structure of BMP-2

107

Figure 3. (a) Topology diagram of BMP-2. Helices are indicated as cylinders, b-strands as arrows. Secondary structure elements which are part of the TGFb/BMP basic-scaffold are colored blue (for details see the text). Orange ones (a1 and a2) are uniquely found in TGF-bs, red ones are unique to BMP-7 (a4), and green ones are unique to BMP-2 (b5a). The secondary structure assignment has been carried out with PROCHECK (Laskowski et al., 1992). (b) Structure-based sequence alignment of TGF-b family proteins. The secondary structure assignment refers to the X-ray structure of BMP-2. Residues that are not visible in the models are indicated as lowercase characters. Residues Ê ). Those located in the ®nger-region of the involved in dimer contacts are conserved (contact cutoff-distance 3.7 A protein are shaded in light gray, dark gray indicates residues located in helix a3. (c) Sketch of the BMP-2 dimer for visualization of the intersubunit angle. Looking at the dimer from the side along the crystallographic 2-fold axis, the molecule was rotated 90  around the longest transmolecular axis (passing the molecule from left to right in the picture) to demonstrate the 40  angle between the subunits.

structure in which the b-strands b2, b5, b6 and b9 are arranged close enough to form a short segment of four-stranded antiparallel b-sheet. This arrangement can also be observed in all known structures of TGF-b family proteins. Additionally, Phe41 is involved in an interchain contact (Phe41Ê ) to the neighboring subunit. O


His60-Ne2 3.2 A Corresponding residues in the other TGF-b-like proteins (BMP-7 Tyr65; TGF-b2 Asn42; TGF-b3

Asn42) also adopt unusual f/c-angles that are imposed by comparable inter- and intrachain contacts. Thus, this conserved feature seems to be crucial for the formation of the interface (Mittl et al., 1996). BMP-2 shows a cis-peptide bond for Pro35 (Figure 2) which is de®ned unambiguously by electron density. The cis-conformation is stabilized by a hydrogen-bond between Ala34-O and Leu90-

108 Ê ). All solved structures of the TGF-b like N (2.71 A proteins show a cis-peptide bond for this invariant proline residue. As stated by Schlunegger & Gruetter (1993) in the case of TGF-b2, no explicit reason for the existence of the cis-conformation can be found. Nevertheless, the conservation in TGF-bs and BMPs implies an important function of this unusual bond that may become clear when a crystal structure of BMP-2 in complex with a receptor molecule is available. A molecule of MPD has been identi®ed in the difference density map. MPD has bound to the protein during soaking the crystal in the cryoprotection solution (see Materials and Methods). Thus, it has occupied an open site and was not involved in crystal formation. MPD binds in the hydrophobic vicinity of Trp28 and Trp31 in ®ngertip 1 loop. Loose hydrophobic contacts are formed between the methyl groups of the ligand and the side-chains of Trp28, Trp31 and Tyr103; one strong hydrogen bond is visible between Asp59-Od1 and Ê ). Its 2the 4-hydroyxl group of MPD (2.9 A hydroxyl group forms a hydrogen bond to a water Ê ). Remarkably, Mittl et al. molecule (Wat32, 3.0 A (1996) found a dioxane molecule in their model of TGF-b3 in a similar position to that of MPD in

Crystal Structure of BMP-2

BMP-2. Possibly, the ®nger-helix cavity (Figure 4) whose bottom is formed by Trp28 and Trp31 represents a hydrophobic pocket which can easily accommodate small amphiphilic molecules like MPD or dioxane. It remains to be elucidated, e.g. by mutational analysis, if this site functions in terms of direct receptor binding or in interaction with receptor carbohydrate side-chains as discussed previously for TGF-b3 (Mittl et al., 1996). BMP-2 produced in animal cells is known to be glycosylated, probably at Asn56 (Israel et al., 1992; Wang et al., 1990). The protein used in the present study has been produced in Escherichia coli and is therefore unglycosylated. The single putative N-glycosylation site at Asn56 (NXT) is situated in the loop N-terminal to helix a3 and it is conserved in the BMPs. BMP-2 made in E. coli has biological activities in vitro (Ruppert et al., 1996) and in vivo (KuÈbler et al., 1998). Thus, glycosylation is not mandatory for its function. Dimer formation Native BMP-2 is a homodimer and its subunits are related by a 2-fold axis. In the crystal one monomer of the protein is the asymmetric unit and

Figure 4. Solvent-accessible surface representation of BMP-2. Red color indicates negative (ÿ10 kT) and blue colour indicates positive electrostatic potential. Hydrophobic regions are colored white (0 kT). The negative potential of the ®nger-helix cavities and the positive charge of central cavity I is clearly visible. The negatively charged central cavity II which is located on the opposite side of the molecule is barely visible. The surface potential has been calculated with Delphi/Grasp (Nicholls et al., 1991).

109

Crystal Structure of BMP-2

hence the dimer is built by crystallographic symmetry. The BMP-2 subunits are covalently linked by a single disul®de bond (Cys78 from both subunits), which is intersected by the 2-fold axis of spacegroup R32. Figure 2 shows the antiparallel association of the dimer. The face-to-face interaction takes place in a way excluding the solventexposed ®ngertips. Intersubunit contacts are formed by interactions of helix a3 with the b-sheets of the neighboring molecule. The same mode of dimerization has been found in the TGF-b proteins and BMP-7, but it is different to more distantly related cystine-knot proteins: PDGF and VEGF dimerize in an antiparallel side-to-side fashion and via two cystine bridges, in NGF the protomers associate parallel and back-to-back, exclusively via non-covalent interactions (McDonald & Hendrickson, 1993; Murray-Rust et al., 1993). A sideview of the dimer perpendicular to the 2-fold crystallographic axis and the top/bottom axis (Figure 3(c)) reveals that the monomers are not assembled in a straight line but with an angle of approximately 40  . As a consequence, the dimerization creates a convex and a concave side on the molecule. A superposition of dimeric BMP2, BMP-7, TGF-b2 and TGF-b3 shows that all proteins share a highly similar core region (cystineknot ‡ b-sheets, Figure 4) and therefore have almost the same intersubunit angles as BMP-2. Reduced structure models (120 residues; N termini, ®ngertip loops and helix a3 deleted) superimpose with the following r.m.s. deviations of the Ca atoms (in parentheses the number of matching Ca: Ê (99), BMP-2/TGF-b2: 1.1 A Ê BMP-2/BMP-7: 1.0 A Ê (83). As the monomers (97), BMP-2/TGF-b3: 1.1 A also superimpose with a high degree of accuracy (Table 2) both the secondary structure and the assembly of the protomers are conserved despite low sequence similarity in the TGF-b superfamily (Table 2). We suggest calling this structurally invariant elementary body ``TGFb/BMP basic scaffold''. Hydrophobic packing is the most abundant form of subunit interactions in the BMP-2 dimer. By means of helix-sheet interactions two tightly packed hydrophobic cores are generated, whereas Table 2. Sequence similarities and r.m.s. deviations of proteins from the TGF-b family TGF-b2 TGF-b2 TGF-b3 BMP-7

TGF-b3

BMP-7

BMP-2

87.50 0.894 (97)

36.04 1.039 (80) 34.23 0.871 (77)

34.26 0.980 (80) 34.86 1.021 (71) 53.51 1.023 (85)

The upper number gives the sequence similarity in % of the given protein pair calculated with GAP (GCG, 1997); the lower Ê 2, the number in number gives the average r.m.s. deviation in A parentheses states the number of matching Ca atoms. r.m.s. deviations have been calculated with BRAGI (Schomburg & Reichelt, 1988).

the isolated monomer by itself has no hydrophobic core. These hydrophobic interactions at the interface are ¯anked by six hydrogen bonds between helix a3 and strand b8 (Asn59Ê ; His60-N


Asp105-O 2.9 A Ê; Nd2


Gln104-O 2.6 A e2 Ê His60-N


Phe41-O 3.2 A; every contact occurs twice). The central region around the intersubunit disul®de bond is less compact and has a more hydrophilic character. In addition, several charged and polar residues (Thr58, Thr82, Asp105, Arg114) as well as water molecules play an important role in the dimer interface. Although only a limited number of water molecules could be reliably identÊ electron density, ten (®ve from i®ed in the 2.7 A each monomer) are involved in hydrogen bonds, linking the subunits (Wat7, -17, -19, -23, -27). They have the lowest B-factors among all water molecules, indicating that they are tightly bound to the dimer interface and hence important for the dimer assembly. A structure-based sequence alignment (Figure 3(b)) shows that the positions and chemical properties of residues which are involved in interchain contacts are highly conserved among TGF-bs and BMPs as reported by Grif®th et al. (1996). The similarity of contact residues is higher than that of the total residues among the four compared sequences: 23 % of the residues are invariant in the complete sequences, but 47 % are conserved among the contact residues. Interestingly, the interacting amino acid residues are not only located in relatively rigid secondary structure elements but also in the ¯exible loop 1 (Val26, Trp28 in BMP-2). The buried surface of each monomer covers an Ê 2 (calculated with DSSP, Kabsch & area of 1380 A Sander, 1983) corresponding to only 12 % of the total solvent-accessible surface. This value is relatively high compared to contact surfaces of antigen-antibody complexes which are typically in Ê 3 (Davies & Cohen, 1996; the range of 600-900 A Janin & Chothia, 1990). A further important contribution to binding energy is provided by the interchain cystine bridge linking both monomers covalently. Indeed, the BMP-2 dimer shows striking stability, as documented in various puri®cation protocols that employ harsh conditions without inactivating the protein (e.g. Sampath & Reddi, 1981; Wozney et al., 1988). Compared to related proteins, BMP-2 shows the largest area of buried surface in the TGF-b superfamily. With identical calculations using the DSSP-program (Kabsch & Ê 2; Sander, 1983) the results are: TGF-b2 1160 A Ê 2. Ê 2; BMP-7 1180 A TGF-b3 1220 A Surfaces and cavities The solvent-accessible surface of the BMP-2 dimer exposes large hydrophobic areas (Figure 5). Especially both sides of the ®ngers show extended non-polar patches. With this observation it becomes clear why BMP-2 has ``unusual solubility properties'' and tends to aggregation as reported by Ruppert et al. (1996). Pronounced charged

110

Crystal Structure of BMP-2

Figure 5. Superposition of TGF-b family proteins. For clarity only monomers of each protein are displayed: BMP-2 in blue, BMP-7 in red, TGF-b2 in orange, TGF-b3 in yellow. The smoothing of the Ca trace was disabled to emphasize the differences in the structure models.

regions are located at the ®ngertips where strong negative potential is visible and in the centre of the molecule where two patches of mixed charge can be observed. Two prominent cavities are discernible at the centre of the protein. Comprising residues from both subunits, cavity I situated on the convex side Ê deep and covers a surface area of the dimer is 10 A Ê  15 A Ê . This cavity has a of approximately 25 A large excess of positive charge: ten of the 12 charged residues in this area have a basic sidechain (Arg9, Lys11, Lys15, Lys73, Lys76, each of them from both subunits). Both N termini fall into the area of cavity I. The eight N-terminal residues of both monomers that were not visible in the electron density comprise an additional ®ve positive charges on each chain. The high concentration of positive charges explains why the N-terminal region (residues 1-17) functions as a heparin binding site (Ruppert et al., 1996). However, it remains unclear to what extent individual positive charges participate in binding of this negatively charged glucosaminoglycan. As heparin is a heterogeneous compound (Lindahl & Kjellen, 1991) the mobility of the BMP-2 N terminus could re¯ect an adaptation towards the variability of the glucosaminoglycan binding partner. Anyhow, the clustering of many positive charges certainly will enhance effective binding. It cannot be rigorously ruled out that cavity I is an artefact, because of the eight missing residues at the N terminus. But under the assumption that the missing region does not fold completely differently as compared to the TGF-bs, it seems very unlikely that it could ®ll the cavity. Furthermore, binding of the positively charged tail would result in strong electrostatic repulsion. In

the TGF-bs the N termini have been completely identi®ed as short helices (Daopin et al., 1992; Mittl et al., 1996; Schlunegger & Grutter, 1993) lying at the edge but facing away from central cavity I. In BMP-7 a similar positively charged cavity I is present. The N terminus, which is 24 residues longer as in BMP-2, is also invisible in the available structure model of BMP-7 (Grif®th et al., 1996). The N termini of related TGF-b proteins show a different composition. Here, negatively charged regions predominate. It is unknown whether a heparin binding site exists in these proteins. The second cavity (cavity II) is located on the concave side of the molecule and is distinctly smalÊ  10 A Ê 5 A Ê ) than cavity I. Again both ler (15 A subunits contribute the same set of residues to cavity II. These are located in the N-terminal loop of helix a3 (Thr58), in b6 (Val80, Pro81, Thr82, Glu83, Leu84), b8 (Asp105) and at the C terminus (Arg114). Negative charges predominate (the acidic side-chains of Glu83 and Asp105 plus the C terminus versus the basic side-chains of Arg114). Cavity II is occupied by several water molecules (Wat13, -17, -19, -23, -27). A similar but less deep cavity is also present in all TGF-bs and in BMP-7. Cavity II of BMP-2 and BMP-7 shows the same charge distribution. The TGF-bs show a very ¯at cavity II and their charge is weakly negative. Remarkably, a dominant negative mutant of zebra®sh BMP-2 (swirl) has a C-terminal extension of six residues (Martinez-Barbera et al., 1997). Cavity II is therefore a possible interaction area for one of the receptor chains. Furthermore, two identical symmetry-related cavities marked ®nger-helix cavity in Figure 5 exist Ê wide and 10 A Ê deep and in BMP-2. They are 18 A

Crystal Structure of BMP-2

are formed by the ®ngertip-loops and parts of the ®ngers from one chain and the C-terminal region of a-helix a3 of the other chain. The outer walls of the cleft show negative potential, on one side from side-chains of the ®ngertips (Asp30, Glu94, Glu96), on the other side from Asp53 of helix a3. The bottom is characterized by mainly hydrophobic residues (Trp28, Trp31, Ile62, Leu66, Tyr103, Met106). As described below, similar, although more shallow ®nger-helix cavities have been found in the related protein structures. The surface potential of the ®nger-helix cavity is different in the TGF and BMP-family, respectively: BMP-2 as well as BMP-7 show an excess of negative charge in the ®ngertip segments, whereas in the TGF-bs a surplus of positive charge is present in this area. The distinctive shape and surface charge pattern of the ®nger-helix cavity in TGF-b-like proteins may suggest that it forms a speci®c epitope for receptor interactions, as previously discussed by Grif®th et al. (1996). Remarkably, in all X-ray structure models of TGF-b superfamily proteins solved to date the ®ngertip-loops and helix a3 which form the borders of the ®nger-helix cavities show elevated B-factors. It remains to be elucidated whether this ¯exibility has any relevance for receptor interaction. Topological similarities in the TGF-b b superfamily On the basis of sequence similarities the TGF-b superfamily is commonly divided into several families: the BMP/OP-family, which comprises the BMP-2/4-subgroup and the BMP-7/OP-subgroup, the activin/inhibin-family, the TGF-b-family and others (Grif®th et al., 1996). Despite their low degree of sequence similarity (around 35 %) TGFbs and BMPs form folding patterns of striking uniformity (Table 2, Figure 4). As described above, superimposed structures show small r.m.s. deviÊ . The protein ations of the Ca atoms of around 1 A core with its b-sheets and the cystine-knot are virtually identical within the different superfamily members. Major differences can be observed in the ®ngertip-loops and helix a3 with its connected loops which have divergent orientations in the respective proteins. This leads to the conclusion that the cystine-knot and the ®ngers formed by b-sheets act as a stable and uniform scaffold for elaboration of loops of relatively low sequence similarity. Corresponding topological observations have been reported for the even more distantly related proteins NGF and VEGF which show a similar monomer fold but a different dimer assembly (McDonald & Hendrickson, 1993; Murray-Rust et al., 1993). In the VEGF/Flt-1 complex (Wiesmann et al., 1997) the loops (residues 46-48, 79-91) analogous to the ®ngertip loops 1 and 2 in BMP-2 provide important residues for ligandreceptor interaction. This supports the hypothesis that receptor speci®city may be generally located in the variable loops.

111 Mittl and co-workers (1996) reported for the TGF-b2/TGF-b3 pair that both ®ngertip-loops when analyzed as isolated units superimpose with good agreement. They de®ned these loops of the TGF-b molecules as rigid units that adopt different orientations in proteins of the TGF-b superfamily. This result, originally found for the TGF-b family, can now be extended to the BMP/OP-family. Superimposing BMP-2 (residues 22-39 and 86-104) with BMP-7 (residues 46-63 and 111-129), the Ê for average r.m.s. deviation of Ca atoms is 0.6 A Ê for the second stretch of resithe ®rst and 0.7 A dues. The same comparison of BMP-2 with TGF-b2 (residues 23-40 and 85-102) reveals r.m.s. deviÊ and 0.9 A Ê , respectively. As can be ations of 0.6 A seen in Figure 5 the BMP-2 ®ngertip 2 loop adopts an orientation different from that of all the other proteins. While these loops extend in the plane of b7 and b8 in BMP-7, TGF-b2, and TGF-b3, in BMP2 it is nearly perpendicular to the b-strands. This different orientation possibly is due to altered crystal contacts (Figure 6), since BMP-7, TGF-b2 and TGF-b3 have been crystallized in spacegroup P3221, whereas BMP-2 crystals belong to spacegroup R32. Contacts of symmetry-related molecules in the ®ngertip region of BMP-2 are formed between residues 96-98 and 100-102, as well as residues 33-35 with the same stretch of residues in the neighbouring molecule. This latter contact region is also present in the P3221 crystal forms (e.g. residues 29-31 in TGF-b3). But the rest of the crystal packing is different: additional interactions are present for example in BMP-7 between residues 34-36 and 116-118, as well as residues 42-44 and 120-123 (Figure 6). Although crystal-packing contacts may induce conformational diversity, the ``frozen'' conformations within crystals have been shown to be true low-energy conformations (Kossiakoff et al., 1992). A triclinic crystal form of VEGF revealed eight different orientations for a speci®c surface loop (Muller et al., 1997a), comparable to ®ngertip loop 2 in the TGF-b superfamily. The ¯exibility of the VEGF surface loop is suggested to be responsible for promiscuity and speci®city of receptor binding. A rigid body shift as observed for the ®ngertip loops can also be detected for helix a3 and the connected loops. Although the helices have clearly different orientations in each of the structure models, the r.m.s. deviations of superimposed helical regions including their adjacent loops are rather small: BMP-2 (residues 49-78)/BMP-7 (residues 73Ê ; BMP-2/TGF-b2 (residues 50-77) 0.5 A Ê; 103) 0.8 A Ê . The most BMP-2/TGF-b2 (residues 50-77) 0.6 A signi®cant differences are located in the loop N-terminal to the helix. This is the result of a three residue insertion compared to the TGF-bs (Figure 3). The interhelical angle of the helices a3 in each of the subunits re¯ects the membership of a speci®c subgroup. The BMPs, belonging to the BMP/OP-subgroup, show an angle of about 48  between the helices (BMP-2 49.1  ; BMP-7 47.4  ), whereas the TGF-bs form an interhelical angle

112

Crystal Structure of BMP-2

Figure 6. Stereoview of the crystal packing. The upper panel shows BMP-2 (spacegroup R32), the lower one BMP-7 (spacegroup P3221). The asymmetric unit is colored in blue, the symmetry-related molecules in black. Fingertip 2 loop whose orientation might be altered by crystal contacts has been colored red (BMP-2 residues 92-100; BMP-7 residues 117-125).

approximately 10  smaller (TGF-b2 37.6  ; TGF-b3 37.3  ). Nevertheless, the general conformation of the ®ngertip loops and helix a3 seems to be conserved. The above results clearly show that the amino acid sequence does not only code for the typical tertiary structure of the TGF-b superfamily with a virtually identical core and three rigid modules that can orient slightly different in space, but it also determines the speci®c mode of dimerization with the result of astonishingly similar three-dimensional structures.

Materials and Methods Crystallization and data acquisition The cloning, sequencing and expression of recombinant human BMP-2 from E. coli has been described (Ruppert et al., 1996). The protein representing the mature part of the precursor (residues 283-396, accession number P12643, Swissprot database, rel. 35, Stoesser et al., 1997) was puri®ed using size-exclusion and cationic exchange chromatography. After dialysis the resulting protein fraction was lyophilized and stored at ÿ20  C. Purity and homogeneity of the protein were checked by electrophoretic techniques and analytical HPLC. The biological activity was tested with a limb bud assay as described by Ruppert et al. (1996). For crystallization, a solution containing 5 mg/ml BMP-2 in 20 mM acetate buffer (pH 3.5) was prepared. Initial crystallization trials were carried out by the screening methods described by Jancarik & Kim (1991) and Cudney et al. (1994). Premixed screening solutions are commercially available as Crystal Screen I and II

(Hampton Research, Laguna Hills, CA). Small crystals were obtained in a 50 %-screen with conditions containing high concentrations of MPD or tert-butanol as precipitant. The crystallization conditions using MPD could not be re®ned, but further trials revealed lithium sulfate to be a necessary co-precipitant. Together with tert-butanol at acidic pH, high quality but very fragile trigonal crystals of spacegroup R32 Ê , c ˆ 107.75 A Ê ) were obtained by the (a ˆ b ˆ 91.44 A hanging drop vapor diffusion method at 20  C. The reservoir solution was composed of 50 mM citrate buffer (pH 5.4), 100 mM lithium sulfate and 12 % (v/v) tertbutanol. The crystallization was introduced by mixing 2 ml of reservoir solution with 2 ml of protein solution. The crystals reached a maximal size of 250 mm  200 mm  100 mm in one to two weeks. Diffraction data were collected from a single crystal at 100 K with a Xentronics area detector on a Rigaku rotating anode. The cryoprotection solution contained 20 mM sodium citrate (pH 5.4), 40 mM lithium sulfate, 5 % (v/v) tert-butanol, Ê , data 18 % MPD. Although the crystal diffracted to 2.4 A Ê were rejected because of high Rmerge values beyond 2.7 A and completeness below 70 %. Data processing was carried out with XDS (Kabsch, 1993). Table 1 gives a summary over the data processing statistics. Structure determination The structure determination was started with the calculation of a self-rotation function. The resulting Patterson map produced by REPLACE (Tong & Rossmann, Ê revealed no non-crystallographic sym1990) at 8.0-4.0 A metry. Under the assumption that one BMP-2 monomer is the asymmetric unit, the Matthews coef®cient Ê 3/Da corresponding to a (Matthews, 1968) VM is 3.0 A solvent content of 59 %.

113

Crystal Structure of BMP-2

Initial phases were calculated by molecular replaceÊ using the monomeric ment at a resolution of 10-3.5 A structure of transforming growth factor b-2 (TGF-b2, PDB-code 1TFG) as a search model, which has 34.3 % sequence identity with BMP-2 (program AMoRe; CCP4, 1994). Despite the fact that BMP-7 (PDB code 1BMP) has a higher sequence similarity to BMP-2 (53.5 %) than TGFb2, we were not able to ®nd a cross-rotation function with good agreement with the former protein as search model. The two best cross-rotation functions calculated in the Ê were related by a 2-fold axis resolution range of 10-3.5 A had a poor correlation coef®cient of 9.4 %, but were clearly separated from the second-best with correlation coef®cient of 8.7 %. The subsequently calculated translation function revealed a correlation coef®cient of 23.3 % and an R-factor of 52.1 %. A ®nal rigid body re®nement (AMoRe) improved both the correlation coef®cient and the R-factor to 35.5 % and 49.0 %, respectively. After selection of 6 % of the re¯exions as test-set for Ê ) and crossvalidation, rigid body re®nement (10-3.5 A Ê ; Brunger, 1989) was persimulated annealing (10-3.0 A formed using XPLOR 3.1 (Brunger, 1990) followed by manual rebuilding using O (Jones et al., 1991). Gradually all side-chains were replaced in the BMP-2 model and re®ned by further simulated annealing. Improvement of the structure was achieved in alternating cycles of manual rebuilding (program O) and re®ning with REFMAC (CCP4, 1994), thereby increasing the resolution in steps Ê . At the beginning overall B-factor re®nement of 0.1 A Ê data water molecules were was applied. Reaching 2.8 A added and individual B-factor re®nement was utilized. This decreased both the R-factor by 3 % and Rfree by 5 %. At this stage the re®nement seems to have converged as the R-values could not be further decreased (R 24.8 %; Rfree 29.8 %). This model was subsequently re®ned using the new program CNS, version 0.5 (Brunger et al., 1998). As with REFMAC, a maximum-likelihood type of re®nement was carried out. The resulting maps were signi®cantly improved, therefore it was possible to additionally identify MPD and Arg9 in the difference electron density map. The ®nal minimization cycle decreased R to 24.2 % and Rfree to 27.9 % (no s cutoff applied). A couple of high peaks in the Fo ÿ Fc map were left uninterpreted as they lie too far away from the protein for reasonable contacts and near one of the 3-fold symmetry axes.

Analytical calculations and used data Surface areas were calculated with DSSP (Kabsch & Ê. Sander, 1983). The program uses a probe radius of 1.4 A Electrostatic calculations were performed with the DELPHI-program included in GRASP (Nicholls et al., 1991). Default values were accepted for the solvent dielectric Ê , ionic strength 0.1 M constant 80, solvent radius 1.4 A Ê . The core of the protein was and ionic radius 2.0 A approximated by using a dielectric constant of 2.0. Interhelical angles were calculated with TOP (Lu, 1996). Structure-based sequence alignments and r.m.s. deviations were calculated with BRAGI (Schomburg & Reichelt, 1988). Coordinates of the discussed proteins were used as deposited in the PDB databank: BMP-7 1BMP (Grif®th et al., 1996); TGF-b2 1TFG (Schlunegger & Grutter, 1992); TGF-b3 1TGJ (Mittl et al., 1996); VEGF 1VPF (Muller et al., 1997b); PDGF 1PDG (Oefner et al., 1992).

Protein Data Bank accession number Coordinates and structure factors have been deposited in the Brookhaven Protein Data Bank (PDB code 2BMP).

Acknowledgements We thank Christian SoÈder for technical assistance as well as Dr Petra Knaus and Dr Matthias Dreyer for helpful discussions and critical reading of the manuscript. This work was supported by the Deutsche Forschungsgesellschaft, grant Se 435/3-2, and by the Fond der Chemischen Industrie.

References Brunger, A. T. (1989). Crystallographic re®nement by simulated annealing. Acta Crystallog. sect. A, 45, 50-61. Brunger, A. T. (1990). Extension of molecular replacement: A new strategy based on Patterson correlation re®nement. Acta Crystallog. sect. A, 46, 46-57. Brunger, A. T. (1997). Free R Value: cross-validation in crystallography. Methods Enzymol. 277, 366-396. Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T. & Warren, G. L. (1998). Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallog. sect. D, 54, 905-921. CCP4, (1994). The CCP4-suite: programs for crystallography. Acta Crystallog. sect. A, 50, 760-763. Carson, M. & Bugg, C. E. (1986). Algorithm for ribbon models of proteins. J. Mol. Graph. 4, 121-122. Celeste, A. J., Iannazzi, J. A., Taylor, R. C., Hewick, R. M., Rosen, V., Wang, E. A. & Wozney, J. M. (1990). Identi®cation of transforming growth factor beta family members present in bone-inductive protein puri®ed from bovine bone. Proc. Natl Acad. Sci. USA, 87, 9843-9847. Chalaux, E., Lopez, Rovira T., Rosa, J. L., Bartrons, R. & Ventura, F. (1998). JunB is involved in the inhibition of myogenic differentiation by bone morphogenetic protein-2. J. Biol. Chem. 273, 537-543. Cudney, B., Patel, S., Weisgraber, K., Newhouse, Y. & McPhearson, A. (1994). Screening and optimization strategies for macromolecular crystal growth. Acta Crystallog. sect. D, 50, 141-423. Daopin, S., Piez, K. A., Ogawa, Y. & Davies, D. R. (1992). Crystal structure of transforming growth factor-beta 2: an unusual fold for the superfamily. Science, 257, 369-373. Davies, D. R. & Cohen, G. H. (1996). Interactions of protein antigens with antibodies. Proc. Natl Acad. Sci. USA, 93, 7-12. Dudley, A. T., Lyons, K. M. & Robertson, E. J. (1995). A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes Dev. 9, 2795-2807. Estevez, M., Attisano, L., Wrana, J. L., Albert, P. S., Massague, J. & Riddle, D. L. (1993). The daf-4 gene encodes a bone morphogenetic protein receptor controlling C. elegans dauer larva development. Nature, 365, 644-649.

114 Genetics Computer Group (1997). Wisconsin Package, Version 9.1, Genetics Computer Group (GCG), Madison, WI. Goetschy, J. F., Letourneur, O., Cerletti, N. & Horisberger, M. A. (1996). The unglycosylated extracellular domain of type-II receptor for transforming growth factor-beta. A novel assay for characterizing ligand af®nity and speci®city. Eur. J. Biochem. 241, 355-362. Grif®th, D. L., Keck, P. C., Sampath, T. K., Rueger, D. C. & Carlson, W. D. (1996). Three-dimensional structure of recombinant human osteogenic protein 1: structural paradigm for the transforming growth factor beta superfamily. Proc. Natl Acad. Sci. USA, 93, 878-883. Hinck, A. P., Archer, S. J., Qian, S. W., Roberts, A. B., Sporn, M. B., Weatherbee, J. A., Tsang, M. L., Lucas, R., Zhang, B. L., Wenker, J. & Torchia, D. A. (1996). Transforming growth factor beta 1: threedimensional structure in solution and comparison with the X-ray structure of transforming growth factor beta 2. Biochemistry, 35, 8517-8534. Hogan, B. L. M. (1996). Bone morphogenetic proteins in development. Curr. Opin. Genet. Dev. 6, 432-438. Israel, D. I., Nove, J., Kerns, K. M., Moutsatsos, I. K. & Kaufman, R. J. (1992). Expression and characterization of bone morphogenetic protein-2 in Chinese hamster ovary cells. Growth Factors, 7, 139-150. Jancarik, J. & Kim, S. H. (1991). Sparse matrix sampling: a screning method for crystallization of proteins. J. Appl. Crystallog. 24, 409-411. Janin, J. & Chothia, C. (1990). The structure of proteinprotein recognition sites. J. Biol. Chem. 265, 1602716030. Jones, T. A., Zou, J. Y., Cowtan, S. W. & Kjelgaard, M. (1991). Improved methods for building protein in electron density maps and location of errors in these models. Acta Crystallog. sect. A, 47, 110-119. Kabsch, W. (1993). Automatic processing of rotation diffraction data from crystals of initially known symmetry and cell constants. J. Appl. Crystallog. 26, 795-800. Kabsch, W. & Sander, C. (1983). Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers, 22, 2577-2637. Kingsley, D. M. (1994). The TGF-beta superfamily: new members, new receptors, and new genetic tests of function in different organisms. Genes Dev. 8, 133146. Koenig, B. B., Cook, J. S., Wolsing, D. H., Ting, J., Tiesman, J. P., Correa, P. E., Olson, C. A., Pecquet, A. L., Ventura, F. & Grant, R. A., et al. (1994). Characterization and cloning of a receptor for BMP2 and BMP-4 from NIH 3T3 cells. Mol. Cell. Biol. 14, 5961-5974. Kossiakoff, A. A., Randal, M., Guenot, J. & Eigenbrot, C. (1992). Variability of conformations at crystal contacts in BPTI represent true low-energy structures: correspondence among lattice packing and molecular dynamics structures. Proteins: Struct. Funct. Genet. 14, 65-74. KuÈbler, N. R., Reuther, J. F., Faller, G., Kirchner, T., Ruppert, R. & Sebald, W. (1998). Inductive properties of recombinant human BMP-2 produced in a bacterial expression system. Int. J. Oral Maxillofacial Surg. 27, 305-309. Laskowski, R. A., MacArthur, M. W., Moss, D. S. & Thornton, M. J. (1992). PROCHECK: a program to

Crystal Structure of BMP-2 check the stereochemical quality of protein structures. J. Appl. Crystallog. 26, 283-291. Lindahl, U. & Kjellen, L. (1991). Heparin or heparan sulfate - what is the difference? Thromb. Haemost. 66, 44-48. Liu, F., Ventura, F., Doody, J. & Massague, J. (1995). Human type II receptor for bone morphogenic proteins (BMPs): extension of the two-kinase receptor model to the BMPs. Mol. Cell. Biol. 15, 3479-3486. Lu, W. (1996). A WWW service system for automatic comparison of protein structures. Protein Data Bank Quart. Newsletter, 78, 10-11. Luo, G., Hofmann, C., Bronckers, A. L., Sohocki, M., Bradley, A. & Karsenty, G. (1995). BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning. Genes Dev. 9, 2808-2820. Martinez, Barbera J. P., Toresson, H., Da Rocha, S. & Krauss, S. (1997). Cloning and expression of three members of the zebra®sh Bmp family: Bmp2a, Bmp2b and Bmp4. Gene, 198, 53-59. Matthews, B. W. (1968). Solvent content of protein crystals. J. Mol. Biol. 33, 491-497. McDonald, N. Q. & Hendrickson, W. A. (1993). A structural superfamily of growth factors containing a cystine knot motif. Cell, 73, 421-424. Mittl, P. R., Priestle, J. P., Cox, D. A., McMaster, G., Cerletti, N. & Grutter, M. G. (1996). The crystal structure of TGF-beta 3 and comparison to TGFbeta 2: implications for receptor binding. Protein Sci. 5, 1261-1271. Miya, T., Morita, K., Suzuki, A., Ueno, N. & Satoh, N. (1997). Functional analysis of an ascidian homologue of vertebrate Bmp-2/Bmp-4 suggests its role in the inhibition of neural fate speci®cation. Development, 124, 5149-5159. Muller, Y. A., Christinger, H. W., Keyt, B. A. & de Vos, A. M. (1997a). The crystal structure of vascular Ê endothelial growth factor (VEGF) re®ned to 1.93 A resolution: multiple copy ¯exibility and receptor binding. Structure, 5, 1325-1338. Muller, Y. A., Li, B., Christinger, H. W., Wells, J. A., Cunningham, B. C. & de Vos, A. M. (1997b). Vascular endothelial growth factor: crystal structure and functional mapping of the kinase domain receptor binding site. Proc. Natl Acad. Sci. USA, 94, 71927197. Murray-Rust, J., McDonald, N. Q., Blundell, T. L., Hosang, M., Oefner, C., Winkler, F. & Bradshaw, R. A. (1993). Topological similarities in TGF-beta 2, PDGF-BB and NGF de®ne a superfamily of polypeptide growth factors. Structure, 1, 153-159. Newfeld, S. J., Padgett, R. W., Findley, S. D., Richter, B. G., Sanicola, M., de Cuevas, M. & Gelbart, W. M. (1997). Molecular evolution at the decapentaplegic locus in Drosophila. Genetics, 145, 297-309. Nicholls, A., Sharp, K. A. & Honig, B. (1991). Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins: Struct. Funct. Genet. 11, 281-296. Oefner, C., D'Arcy, A., Winkler, F. K., Eggimann, B. & Hosang, M. (1992). Crystal structure of human platelet-derived growth factor BB. EMBO J. 11, 3921-3926. Ozkaynak, E., Rueger, D. C., Drier, E. A., Corbett, C., Ridge, R. J., Sampath, T. K. & Oppermann, H. (1990). OP-1 cDNA encodes an osteogenic protein in the TGF-beta family. EMBO J. 9, 2085-2093.

Crystal Structure of BMP-2 Plessow, S., Koster, M. & Knochel, W. (1991). cDna sequence of Xenopus laevis bone morphogenetic protein 2 (Bmp-2). Biochim. Biophys. Acta, 1089, 280-282. Ramachandran, G. N. & Sasisekharan, V. (1968). Conformation of polypeptides and proteins. Advan. Protein Chem. 23, 283-438. Reddi, A. H. (1997). Bone morphogenetic proteins: an unconventional approach to isolation of ®rst mammalian morphogens. Cytokine Growth Factor Rev. 8, 11-20. Ruppert, R., Hoffmann, E. & Sebald, W. (1996). Human bone morphogenetic protein 2 contains a heparinbinding site which modi®es its biological activity. Eur. J. Biochem. 237, 295-302. Sampath, T. K. & Reddi, A. H. (1981). Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation. Proc. Natl Acad. Sci. USA, 78, 7599-7603. Schlunegger, M. P. & Grutter, M. G. (1992). An unusual Ê feature revealed by the crystal structure at 2.2 A resolution of human transforming growth factorbeta 2. Nature, 358, 430-434. Schlunegger, M. P. & Grutter, M. G. (1993). Re®ned crystal structure of human transforming growth factor beta 2 at 1.95 A resolution. J. Mol. Biol. 231, 445-458. Schomburg, D. & Reichelt, J. (1988). BRAGI: a comprehensive protein modelling system. J. Mol. Graph. 6, 161-165. Sibanda, B. L. & Thornton, J. M. (1991). Conformation of beta hairpins in protein structures: classi®cation and diversity in homologous structures. Methods Enzymol. 202, 59-82. Stoesser, G., Sterk, P., Tuli, M. A., Stoehr, P. J. & Cameron, G. N. (1997). The EMBL Nucleotide Sequence Database. Nucl. Acids Res. 25, 7-14. ten Dijke, P., Yamashita, H., Ichijo, H., Franzen, P., Laiho, M., Miyazono, K. & Heldin, C. H. (1994a). Characterization of type I receptors for transform-

115 ing growth factor-beta and activin. Science, 264, 101-104. ten Dijke, P., Yamashita, H., Sampath, T. K., Reddi, A. H., Estevez, M., Riddle, D. L., Ichijo, H., Heldin, C. H. & Miyazono, K. (1994b). Identi®cation of type I receptors for osteogenic protein-1 and bone morphogenetic protein-4. J. Biol. Chem. 269, 1698516988. Tong, L. & Rossmann, M. G. (1990). The locked rotation function. Acta Crystallog. sect. A, 46, 783-792. Urist, M. R. (1965). Bone: formation by autoinduction. Science, 150, 893-899. Wang, E. A., Rosen, V., D'Alessandro, J. S., Bauduy, M., Cordes, P., Harada, T., Israel, D. I., Hewick, R. M., Kerns, K. M. & LaPan, P., et al. (1990). Recombinant human bone morphogenetic protein induces bone formation. Proc. Natl Acad. Sci. USA, 87, 2220-2224. Wiesmann, C., Fuh, G., Christinger, H. W., Eigenbrot, C., Wells, J. A. & de Vos, A. M. (1997). Crystal structure at 1.7 A resolution of VEGF in complex with domain 2 of the Flt-1 receptor. Cell, 91, 695704. Wozney, J. M., Rosen, V., Celeste, A. J., Mitsock, L. M., Whitters, M. J., Kriz, R. W., Hewick, R. M. & Wang, E. A. (1988). Novel regulators of bone formation: molecular clones and activities. Science, 242, 15281534. Wrana, J. L., Attisano, L., Wieser, R., Ventura, F. & Massague, J. (1994). Mechanism of activation of the TGF-beta receptor. Nature, 370, 341-347. Yamashita, H., ten Dijke, P., Huylebroeck, D., Sampath, T. K., Andries, M., Smith, J. C., Heldin, C. H. & Miyazono, K. (1995). Osteogenic protein-1 binds to activin type II receptors and induces certain activinlike effects. J. Cell Biol. 130, 217-226. Zhang, H. & Bradley, A. (1996). Mice de®cient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. Development, 122, 29772986.

Edited by R. Huber (Received 24 August 1998; received in revised form 25 January 1999; accepted 25 January 1999)