The clip-segment of the von Willebrand domain 1 of the BMP modulator protein Crossveinless 2 is preformed.

Juliane E Fiebig,Thomas D Mueller,Li-Yan Qiu,Hartmut Oschkinat,Markus Bauer,Walter Sebald,Stella E Weidauer,Monika Beerbaum,Jin-Li Zhang,Peter Schmieder

doi:10.3390/molecules181011658

Abstract

Bone Morphogenetic Proteins (BMPs) are secreted protein hormones that act as morphogens and exert essential roles during embryonic development of tissues and organs. Signaling by BMPs occurs via hetero-oligomerization of two types of serine/threonine kinase transmembrane receptors. Due to the small number of available receptors for a large number of BMP ligands ligand-receptor promiscuity presents an evident problem requiring additional regulatory mechanisms for ligand-specific signaling. Such additional regulation is achieved through a plethora of extracellular antagonists, among them members of the Chordin superfamily, that modulate BMP signaling activity by binding. The key-element in Chordin-related antagonists for interacting with BMPs is the von Willebrand type C (VWC) module, which is a small domain of about 50 to 60 residues occurring in many different proteins. Although a structure of the VWC domain of the Chordin-member Crossveinless 2 (CV2) bound to BMP-2 has been determined by X-ray crystallography, the molecular mechanism by which the VWC domain binds BMPs has remained unclear. Here we present the NMR structure of the Danio rerio CV2 VWC1 domain in its unbound state showing that the key features for high affinity binding to BMP-2 is a pre-oriented peptide loop.

Highlights

Bone morphogenetic proteins (BMPs) are secreted protein hormones, which form a subgroup within the large Transforming Growth Factor-β (TGF-β) superfamily
VWC1 to VWC5), each of them between 50 to 60 residues long, and with the first von Willebrand type C (VWC) domain directly starting after the signal peptide (D. rerio CV2 VWC1: 9–64; VWC2: 67–122; VWC3: 125–183; VWC4: 197–246; VWC5: 258–314)
The determination of the structure of CV2 VWC1 in its unbound conformation using NMR spectroscopy highly complements the data provided by the crystallographic study

Summary

Introduction

Bone morphogenetic proteins (BMPs) are secreted protein hormones, which form a subgroup within the large Transforming Growth Factor-β (TGF-β) superfamily (for recent reviews see [1,2]). A mutation of proline 35 in Noggin, which is the residue in the Clip segment mimicking the hydrophobic knob-into-hole interaction of BMP-type I receptor interaction, to serine or arginine has been found in patients suffering from skeletal malformation disorders suggesting a loss of Noggin function likely due to loss of BMP binding [32,33,34] This raises the question how the Clip segment can become a major contributor to the overall binding energy for interacting with BMP ligands, if we assume that due to amino acid sequence and lack of interaction with of this region with the structured rest of Noggin or CV2 VWC1 domain this short peptide stretch adopts its proper conformation just upon binding to the BMP ligand. To test the latter hypothesis we have determined the structure of the VWC1 domain of Crossveinless 2 of Danio rerio by NMR spectroscopy in its free unbound state

Results and Discussion

The Clip Segment Must be Pre-Oriented for High Affinity Binding to BMPs

Expression and Purification of the VWC1 Domain of Crossveinless 2

NMR Data Acquisition and Analysis

In Vitro Interaction Analysis Using Surface Plasmon Resonance

Conclusions