Supplementary material to:

Sequential resonance assignment of the human BMP type II receptor

extracellular domain

Biological context

Bone morphogenetic proteins (BMPs) are members of the transforming growth

factor-β (TGF-β) superfamily (Ozkaynak et al., 1990). They play key roles in many

developmental processes as well as in the regulation of apoptosis. BMPs and other signal

ligands of the TGF-β superfamily, such as TGF-βs, growth and differentiation factors

(GDFs), and activins, exert their biological effects by binding and bringing together two

structurally similar, single-pass transmembrane receptors, classified as type I and II. The

five type II receptors of the superfamily that have been identified have been shown to

differ in terms of their ligand binding specificity. The TGF-β type II receptor (TβR-II),

for example, has been shown to be highly specific for binding TGF-β ligands, whereas

others, such as the activin and BMP type II receptors (ActR-II and BMPR-II,

respectively) have been shown to bind a broad, but non-overlapping range of ligands,

including activins, BMPs and GDFs.

To better understand the interactions that determine specificity of ligand-type II

receptor pairings, and to complement the existing structural information for the ActR-II

(Greenwald et al., 1999; Gray, et al., 2000) and TβR-II (Hinck, et al., 2000) extracellular

domain and the corresponding ligand complexes (Greenwald et al., 2003; Hart, et al.,

2002), we sought to structurally characterize the BMPR-II extracellular domain

(ecBMPR-II) and to study its interactions with BMP and GDF ligands. The results

reported here include the backbone and sidechain resonance assignments of ecBMPR-II.

Methods and results

The ecBMPR-II used in the present study corresponds to the entire predicted

extracellular domain (A26-T150) (Rosenzweig et al., 1995). The ecBMPR-II protein was

expressed with an N-terminal thioredoxin tag and a C-terminal hexahistidine tag in E.

coli strain BL21(DE3). The fusion protein was partially purified by immobilized metal

affinity chromatography and then cleaved with thrombin to yield a final recombinant

product 142 residues in length, including four residues on the N-terminus and thirteen

residues on the C-terminus derived from the vector. The recombinant protein was then

purified to homogeneity by sequential fractionation using high-resolution ion-exchange

and C18 reverse phase chromatography methods. Two types of isotope labeled proteins

were used in the current study, an 15N single-labeled one prepared by culturing the cells

on minimal medium supplemented with 15N-NH4Cl and an 15N, 13C double-labeled one

prepared by culturing the cells on minimal medium supplemented with 15N-NH4Cl and

13C-glucose.

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NMR samples of ecBMPR-II were in 25 mM Na phosphate, 25 mM NaCl, and

5% 2H2O, pH 5.8. The final concentration of the NMR sample was 0.18 mM for 15N

single-labeled ecBMPR-II and 0.3 mM for 15N, 13C double-labeled ecBMPR-II. The

sequence specific 1HN, 15 NH, 13Cα, 13Cβ, and 13C´ assignments of the ecBMPR-II were

made based on a series of sensitivity-enhanced triple-resonance experiments, including

HNCACB, CBCA(CO)NH, HN(CA)CO, and HNCO (Bax, 1994). The side-chain 13C

assignments were made using the C(CO)NH experiment. Backbone Hα and sidechain Hβ

resonances were obtained using the HNHA and HNHB experiments. The other sidechain

1H resonances were assigned using the HCCH-total correlation (TOCSY) experiment

(Bax et al., 1994). All NMR experiments were performed on Bruker Avance 600 and 700

MHz NMR spectrometers at a temperature of 38 ˚C.

Extent of assignments and data deposition

The backbone atoms of 112 of the 125 amino acid residues of ecBMPR-II were

assigned based on the strategy described above (see assigned 1H, 15 N HSQC shown in

Figure 1A). The missing residues include Y67, K81, G83, V100-V101, P105, V125-

T128, P132-P133, and P141. Among the thirteen unassigned residues, partial assignments

are available for five (Y67, K81, G83, V101, and T128). All the unassigned non-prolines

are near cysteines, and eight of the unassigned residues (K81, G83, V100, V101, and

V125-T128) overlap or are near the unassigned residues of human (Hinck et al., 2000) or

chick (Marlow et al., 2000) TβR-II extracellular domain. Such observations suggest that

a common mechanism, possibly isomerization of one or more of the disulfide bonds,

might underlie the missing signals. The four prolines were not assigned because they

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precede other prolines in ecBMPR-II. The chemical shift data were used to analyze the

secondary structure of the ecBMPR-II (Figure 1B). The results show that the protein is

comprised entirely of β-sheet. This is consistent with our expectations based on the X-ray

structure of the most closely related type II receptor whose structure is known, ecActR-II

(Greenwald et al., 1999), as shown (Figure 1B). The assignments have been deposited in

BioMagResBank under accession 6582.

Acknowledgements

This study was supported by Stryker Biotech (J.C.L) and the NIH (GM58670 and

RR13879 to A.P.H. and CA54174 to the macromolecular structure shared resource of the

San Antonio Cancer Institute).

Reference

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Greenwald, J., Fischer, W.H., Vale, W.W. and Choe, S., (1999) Nat. Struct. Biol., 6, 18-

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Greenwald, J., Groppe, J., Gray, P., Wiater, E., Kwiatkowski, W., Vale, W., and Choe, S.

(2003) Mol. Cell., 11, 605-617.

Hart, P.J., Deep S., Taylor A.B., Shu Z., Hinck C.S., Hinck A.P., (2002) Nature Struct.

Biol., 9,203-208.

Hinck, A.P., Walker, K.P. III, Martin, N.R., Deep, S., Hinck, C.S., and Freedberg, D.I.

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(2000) J. Biomol. NMR, 18, 369-370.

Marlow, M.S., Chim, N., Brown, C.B., Barnett, J.V., and Krezel, A.M. (2000) J. Biomol.

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Ozkaynak, E., Rueger, D.C., Drier, E.A., Corbett, C., Ridge, R.J., Sampath, T.K., and

Oppermann, H. (1990) EMBO. J., 9, 2085-2093.

Rosenzweig B.L., Imamura T., Okadome T., Cox G.N., Yamashita H., ten Dijke P.,

Heldin C.H., and Miyazono K. (1995) Proc. Natl. Acad. Sci. U. S. A., 92, 7632-7636.

Wishart, D.S. and Sykes, B.D. (1994) J. Biomol. NMR, 4, 171-180.

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Figure 1. A summary of the backbone assignments and secondary structure of ecBMPR-

II. (A) The 2D 1H-15N HSQC spectrum of E. coli recombinant ecBMPR-II with peaks

labeled according to their assignments. Residues A26-T150 correspond to the fully

predicted extracellular domain of ecBMPR-II (Rosenzweig et al., 1995). Residues G22-

T25 and K151-H163 correspond to vector derived sequence. L139(1) and L139(2) and

S140(1) and S140(2) correspond to pairs of peaks assigned to the same residue. This may

be due to different conformational states that arise due to proline cis:trans isomerization

of one of the two adjacent prolines (P138 or P141). (B) The predicted secondary structure

of the ecBMPR-II domain as deduced by the consensus chemical shift index (CSI)

(Wishart and Sykes, 1994). As indicated by the bold arrows above the CSI plot, the

identity and location of the secondary structures predicted by the CSI method are in good

agreement with those based upon alignment of the primary sequences of ecBMPR-II and

ecActR-II and the X-ray structure of ecActR-II (Greenwald, 1999).

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