Mapping the Deltex binding surface on the Notch ankyrin domain using analytical ultracentrifugation

摘要

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 (NICD), which enters the nucleus and assembles transcriptional activation complexes that drive expression of Notch-responsive genes. The most conserved region of NICD 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, and also a weaker Nank self-association, using sedimentation velocity analytical ultracentrifugation (AUC). 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 three and four as key for mediating heteroassociation. An alanine scan across the interaction loops of Nank identify a conserved hot-spot in repeats three and four, centered at R127, as critical for Dx binding. In addition, we were able to detect weak but reproducible Nank homodimerization (Kd in the mM 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 Deltex interaction appear to be compatible with teterameric Notch2:Dx2 assembly.