Crystallization and preliminary X-ray analysis of the complex of the first von Willebrand type C domain bound to bone morphogenetic protein 2

Abstract

Crossveinless 2 (CV2) is a member of the chordin family, a protein superfamily that modulates the activity of bone morphogenetic proteins such as BMP2. The BMPs represent a large group of secreted proteins that control many steps during embryonal development and in tissue and organ homeostasis in the adult organism. The gene encoding the first von Willebrand type C domain (VWC1) of CV2 was cloned, expressed in Escherichia coli and purified to homogeneity. The binary complex of CV2 VWC1 and BMP2 was purified and subjected to crystallization. Crystals of SeMet-labelled proteins were obtained in two different forms belonging to the tetragonal space groups P4 1 2 1 2 and I4 1 , with unit-cell parameters a = b = 86.7, c = 139.2 Å and a = b = 83.7, c = 139.6 Å , respectively. Initial analysis suggests that a complete binary complex consisting of one BMP2 dimer bound to two CV2 VWC1 domains is present in the asymmetric unit.

The Clip-Segment of the von Willebrand Domain 1 of the BMP Modulator Protein Crossveinless 2 Is Preformed

“…Thus an allosteric effect or influence on the binding mode can be ruled out and the isolated CV2 VWC1 domain can serve as a valid model for the structural and functional interaction of full-length CV2 with BMPs. For structure determination the VWC1 domain comprising residues Leu1 to Gly66 of the mature Danio rerio Crossveinless 2 (UniProt identifier Q5D734) was expressed as a fusion protein to thioredoxin [36]. This approach together with the use of the special E. coli strain Origami that has a deletion of the genes for glutathione reductase ( gor ) and thioredoxin reductase ( trxB ) allows for disulfide bond formation within the cytoplasm of E. coli .…”

“…This approach together with the use of the special E. coli strain Origami that has a deletion of the genes for glutathione reductase ( gor ) and thioredoxin reductase ( trxB ) allows for disulfide bond formation within the cytoplasm of E. coli . As the highly conserved disulfide bonds in VWC domains are important for structural stability and folding, this approach allowed production of functional protein without refolding [36]. However, the strain Origami does not carry the ahpC* mutation required in the gor / trxB double mutant background to allow reduction of essential substrates for the ribonucleotide reductase RNR [37,38] and thus Origami cells did not grow in M9 minimal medium required for isotope labeling.…”