Cystine knots consist of three intertwined disulfide bridges and are considered major determinants of protein stability in proteins in which they occur. We questioned this function and observed that removal of individual disulfide bridges in human vascular endothelial growth factor (VEGF) does not reduce its thermodynamic stability but reduces its unexpected high thermal stability of 108°C by up to 40°C. In wild-type VEGF (⌬G u . A detailed crystallographic analysis of the mutant structures suggests a role of the cystine knot motif in protein folding rather than in the stabilization of the folded state. When assuming that the sequential order of the disulfide bridge formation is conserved between VEGF and glycoprotein ␣-subunit, the crystal structure of the mutant C61A-C104A, which deviates by a root mean square deviation of more than 2.2 Å from wild-type VEGF, identifies a true folding intermediate of VEGF.The cystine knot structural motif consists of three highly intertwined disulfide bridges and occurs in the small inhibitor cystine knot proteins, the cyclotides and the growth factor cystine knot proteins. The first two classes of proteins consist of small polypeptides ranging from 25 to 30 amino acids with functions as diverse as protease inhibition (squash family of inhibitors), ion channel blocking (toxins), and antimicrobial activity (cyclotides) (1-3). The third class consists of the growth factor cystine knot proteins, with members such as transforming growth factor-␤, ␣-nerve growth factor, platelet-derived growth factor, vascular endothelial growth factor (VEGF), 1 bone morphogenic proteins, IL-17s, and many others. These physiologically important growth factors share a common monomer structure but differ in their mode of dimerization (3), thus introducing molecular diversity into the structural mechanism by which these growth factors activate their cognitive receptors (4). Whole genome analyses hint that the cystine knot motif might be very common in extracellular signaling molecules (5).The molecular function of the cystine knot motif is poorly understood. In this motif, two disulfide bridges connect two neighboring chain segments and form a ring structure; a third disulfide bridge penetrates this ring segment and cross-links two additional chain segments. By implication, the cystine knot motif interlocks four separate chain segments. Considerations on the small size of the inhibitor cystine knot proteins and the lack of extended hydrophobic core regions in both the inhibitor and growth factor cystine knot proteins lead to the assumption that this structural motif is a major determinant for the thermodynamic stability of these proteins. Thus, amide exchange experiments estimated the thermodynamic stability of the toxin -MVIIA to be about 4 kcal mol Ϫ1 (6). This equals the stabilization often introduced by single disulfide bridges of 3-5 kcal mol Ϫ1 (7, 8), and therefore it is plausible that removal of single cystines impairs the stability of these proteins.We challenged this assumption and studied t...

The receptor Tyro3 together with Axl and Mer form the Axl/Tyro3 family of receptor tyrosine kinases. Members of this family play essential roles in spermatogenesis, immunoregulation, and phagocytosis. Gas6, the product of growth arrest-specific gene, activates the kinase activity of all three receptors. Here, we report the first biochemical and structural characterization of a member of this family, namely of a fragment spanning the two N-terminal Ig domains of the extracellular part of human Tyro3. Its ligand binding specificity profile is identical to the activation profile of the native receptor. The 1.95-Å crystal structure suggests a common ligandbinding site in this receptor family located at the interface of the Ig domains and unusually rich in cis-prolines. Furthermore, both in the crystal and in solution we observed the ligand-independent dimerization of the receptor fragment. This homophilic interaction emphasizes previous functional reports, which hinted that in addition to signal transduction, members of this family of receptors might participate in cell adhesion.

The production of recombinant proteins in Escherichia coli often leads to the formation of inclusion bodies. Although this has a number of advantages, a major disadvantage is the need to develop folding protocols for the renaturing of the proteins. However, the systematic screening of folding conditions is often hampered by the lack of convenient assays to detect correctly folded proteins. To address this problem we present a simple protocol, which combines folding screens and limited proteolysis to rapidly assess and optimize folding conditions. The efficacy of this method, termed FSAP (folding screening assayed by proteolysis), is demonstrated by the large-scale folding, purification and crystallization of various cystine deletion mutants of the cystine knot family member: vascular endothelial growth factor (VEGF). These mutants are particularly difficult to fold as the cystine knot is believed to make major contributions to the stability of the protein and this family of proteins lacks extensive hydrophobic core regions.