A structural and thermodynamic description of the interaction between deltex and the notch ankyrin domain.

摘要

The evolutionarily conserved Notch signal transduction pathway controls cell fate determination in a broad distribution of tissues during metazoan development. The Notch gene encodes a transmembrane receptor that is cleaved upon binding a transmembrane ligand from an adjacent cell. This cleavage liberates the Notch intracellular domain, which travels to the nucleus and assembles transcriptional activator complexes that drive expression of Notch-responsive genes.;The Notch ankyrin domain (Nank) contains seven ankyrin repeats and makes important contacts in the pathway. Some of the residues important for the formation of transcription factor complexes with CSL and mastermind are within this domain. Additional Nank residues are involved in the interaction between adjacent transcription factor complexes bound to sequence-paired binding sites on DNA. Nank also interacts with the cytosolic protein deltex, although the downstream effects of Nank:deltex binding are still largely unknown. While it has been shown that deltex binds Nank inside the cell, the energetics and structural features of this interaction have not been determined.;In this thesis, I quantified and structurally dissected both Nank:deltex heteroassociation and Nank self-association using sedimentation velocity analytical ultracentrifugation. By directly fitting sedimentation boundaries using SEDANAL, I determined that Nank residue R107 contributes to a weak but functionally relevant Nank self-association with a Kd in the mM range. Through studying the interaction of Nank deletion constructs with deltex, I identified repeats three and four as the most important repeats for mediating heteroassociation. Experiments performed with Nank amino acid substitutions identified R127, a conserved surface residue on the canonical binding face, as critical for binding deltex.;Using Nuclear Magnetic Resonance spectroscopy, I identified residues in the deltex WWE2 domain that are impacted by binding to Nank. Through triple-resonance experiments on 2H-, 15N-, 13C-labeled samples, I assigned 89% of non-proline residues in the structured region of the deltex WWE2 domain. TROSY spectra of deltex WWE2 were obtained for an R107A titration series, and chemical shift changes of the assigned resonances were monitored. Analysis of these spectra revealed a putative binding patch on the N-terminal WWE module that binds to the region of Nank identified by the analytical ultracentrifugation experiments.