Von Willebrand Factor Type C Domain-containing Proteins Regulate Bone Morphogenetic Protein Signaling through Different Recognition Mechanisms

Jin-Li Zhang,Yi Huang,Li-Yan Qiu,Joachim Nickel,Walter Sebald

doi:10.1074/jbc.m700456200

Abstract

Bone morphogenetic protein (BMP) function is regulated in the extracellular space by many modulator proteins, including those containing a von Willebrand factor type C (VWC) domain. The function of the VWC domain-containing proteins in development and diseases has been extensively studied. The structural basis, however, for the mechanism by which BMP is regulated by these proteins is still poorly understood. By analyzing chordin, CHL2 (chordin-like 2), and CV2 (crossveinless 2) as well as their individual VWC domains, we show that the VWC domain is a versatile binding module that in its multiple forms and environments can expose a variety of binding specificities. Three of four, two of three, and one of five VWCs from chordin, CHL2, and CV2, respectively, can bind BMPs. Using an array of BMP-2 mutant proteins, it can be demonstrated that the binding-competent VWC domains all use a specific subset of BMP-2 binding determinants that overlap with the binding site for the type II receptors (knuckle epitope) or for the type I receptors (wrist epitope). This explains the competition between modulator proteins and receptors for BMP binding and therefore the inhibition of BMP signaling. A subset of VWC domains from CHL2 binds to the Tsg (twisted gastrulation) protein similar to chordin. A stable ternary complex consisting of BMP-2, CHL2, and Tsg can be formed, thus making CHL2 a more efficient BMP-2 inhibitor. The VWCs of CV2, however, do not interact with Tsg. The present results show that chordin, CHL2, and CV2 regulate BMP-2 signaling by different recognition mechanisms.

Highlights

Bone morphogenetic proteins (BMPs)2 are secreted growth factors of the TGF-␤ superfamily, which play important roles during embryonic development in pattern formation and tissue specification [1]
We show in this study that the BMP-2 binding epitopes for chordin and CV2 are mainly overlapping with the knuckle epitope of BMP-2, CHL2 binds to both the wrist and knuckle epitopes of BMP-2, and Tsg binds only to the wrist epitope of BMP-2 for type I receptor
To understand the recognition mechanisms of von Willebrand factor type C (VWC) domain-containing proteins for BMPs, we analyzed in detail the binding affinities and specificities of BMP-2 for chordin, CHL2, CV2, some of their VWC domains, and Tsg and identified the binding epitopes of BMP-2 for the modulator proteins

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—All of the modulator proteins and VWC domains were expressed with a C-terminal thrombin cleavage site (LVPRGS) plus a His tag in SF9 insect cells according to the manufacturer’s instructions (PharMingen). CHL2 VWC domains were expressed as follows: 7-VWC1-15, 15-VWC2-20, and 20-VWC3-16 (the numbers indicate numbers of residues in flanking sequences; see supplemental Fig. 1). KD values or rate constants koff/kon were evaluated from one experiment determined for 6 –9 different concentrations of the analytes. The modulator proteins containing His tag were incubated with BMP-2 in 1 ml of binding buffer, followed by 2–5 ␮g/ml anti-His tag antibody (Invitrogen). The complex precipitated with 20 ␮l of protein A-Sepharose beads (Amersham Biosciences) was subjected to SDS-gel electrophoresis under reducing conditions, and BMP-2 was detected with anti-BMP-2 monoclonal antibody (R&D). Inhibition of BMP-2-induced ALP activity by modulators was assessed by incubating C2C12 cells with different concentrations of modulators plus 10 nM BMP-2. Results are given as mean values from six determinations done in parallel for each condition

RESULTS

Apparent KD Chordin

DISCUSSION

VWC sequences on in vivo Sog