Structural prediction of human WNT-5A and Foxy-5

WNT-5A plays critical roles in the health and disease states. For instance, aberrant WNT-5A signaling is associated with several human pathologies such as cancer and inflammation.

The design of small molecules that could mimic some of the molecular functions of WNT-5A was carried out over 10 years ago in collaboration with the group of Prof. Andersson. At that time, the design was guided by several bioinformatics computations that were performed in the absence of 3D structural information about the protein (i.e., the 3D structure of this protein family was not known and thus other types of computations were performed in an attempt to infer structural knowledge from 2D data such as the amino acid sequence). Based upon our in silico predictions, short peptides, expected to be solvent exposed at the surface of WNT-5A, were synthesized and tested experimentally. One such peptide, named Foxy-5, mimicking some functions of WNT-5A, was reported in 2006 by Safholm et al., (J Biol Chem, 281:2740-9). This modified peptide is now in clinical trial. Foxy-5 was expected to be not only solvent exposed but also located in a main protein-protein interaction site (or in a protein-protein interaction exosite depending the definition of site and exosite) and as such should contain amino-acid residues that are important for molecular recognition, the so-called hotspot residues.

The experimental structure of human WNT-5A is not known but today it is possible to build a structural model of WNT-5A using related known X-ray structures. Indeed, it is possible to model in 3D 95% of the amino acids of WNT-5A at a very high confidence level. Mapping Foxy-5 onto the predicted 3D structure of WNT-5A shows that the short peptide segment is indeed essentially solvent exposed (in red on the figure). The peptide could fold as a short loop and alpha helical structure. It is located in an exosite that has been suggested to play a role in protein-protein interactions in this family of signaling molecule (possibly interactions with co-receptors and/or acting on dimerization and therefore could act on modulating the signal by the main receptors...). Mechanistically, the position of Foxy-5 in 3D on the surface of WNT-5A is compatible with a WNT-5A-(co-)receptor interaction site (and/or dimerization site/self assembly site...) and it is indeed in a region predicted to contain hotspot residues as computed using a special type of probe (fragment) mapping algorithm (this approach analyzes the energetics of the WNT-5a surface and the presence of small cavities) (inset figure: orange, yellow, green and blue spheres). This type of computed hotspots should be considered here as regions where a ligand (small or large) could bind. Other hotspot zones, computed with another type of method that take into account sequence conservation and 3D shape of WNT-5A point to several areas including the two protruding loops, at the bottom of this figure (in this orientation). These two zones form a key interaction site for the binding of some receptors in this family of signalling molecules. Additional analysis of all these "binding zones" is ongoing.

In summary, Foxy-5 could adopt an essentially helical plus "loop-like" structure in solution, a 3D structural motif often involved in protein-protein interactions. This segment is solvent exposed and could be involved in macromolecular interactions in full length WNT-5A. Foxy-5 is not located in the main protein-protein interaction site known in this family (the main site involves two protruding loops at the bottom of the figure in this orientation) but is located in an exosite expected to be critical for interactions with WNT-5A some receptor(s) and/or co-receptor(s) and/or be part of a dimerization site. Such observations are compatible with the fact that Foxy-5 mimics some of the molecular functions of full length WNT-5A. These computations should help to gain new insights into the structure and function of WNT-5A and Foxy-5. Additional computations are ongoing and the results will be published in the coming months in an attempt to understand the molecular events at the atomic level.