Mapping the Deltex-Binding Surface on the Notch Ankyrin Domain Using Analytical Ultracentrifugation

The Notch signal transduction pathway controls cell fate determination during metazoan development. The Notch gene encodes a transmembrane receptor that is cleaved upon activation, liberating the Notch intracellular domain, which enters the nucleus and assembles transcriptional activation complexes that drive expression of Notch-responsive genes. The most conserved region of the Notch intracellular domain is an ankyrin domain (Nank), which binds directly to the cytosolic effector protein Deltex (Dx), controlling intracellular Notch activity. However, the structural and energetic basis for this interaction remains unknown.Here, we analyze the thermodynamics and hydrodynamics of the Nank:Dx heteroassociation, as well as a weaker Nank self-association, using sedimentation velocity analytical ultracentrifugation. By comparing g(s⁎) and c(s) distributions, and by direct fitting of sedimentation boundaries with thermodynamic association models, we were able to characterize the Nank:Dx heterodimer, measure its affinity, and map the interaction on the surface on Nank. N- and C-terminal deletions of whole ankyrin units implicate repeats 3 and 4 as key for mediating heteroassociation. An alanine scan across the interaction loops of Nank identifies a conserved hot spot in repeats 3 and 4, centered at R127, as critical for Dx binding. In addition, we were able to detect weak but reproducible Nank homodimerization (Kd in the millimolar range). This association is disrupted by substitution of a conserved arginine (R107) with alanine, a residue previously implicated in a functionally relevant mode of interaction within dimeric transcription complexes. The distinct binding surfaces on Nank for homotypic versus Dx interaction appear to be compatible with teterameric Notch2:Dx2 assembly.

Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (105 K)Download as PowerPoint slideHighlights► Associations of the Notch ANK domain and Dx WWE2 domain are analyzed by sedimentation velocity analytical ultracentrifugation. ► Both domains undergo weak homotypic associations (Kd approximately in the millimolar range). ► Homodimerization of the Notch ANK domain is disrupted by the substitution R107A. ► Deletion and substitution studies of Notch ANK map Dx binding to repeats 3 and 4. ► Notch ANK:Dx interaction overlaps CSL, but may tetramerize.