Intrinsic Global Disorder and Inducible Local Order in the Cytoplasmic Tail of the Notch Ligand Delta-like 4

Signaling mediated by Notch receptors and their ligands is essential in cell differentiation and morphogenesis in metazoans. As both receptors and ligands are cell-surface expressed proteins, Notch signaling is restricted to nearby interacting cells. The five human ligands of Notch receptors are all single-pass, type I transmembrane proteins consisting of an extracellular region involved in receptor binding and of a 100-150 residue intracellular tail. One of these ligands, Delta-like 4 (DLL4) is a human homologue of Drosophila Delta protein, and plays an important role in the development of blood vessels. The intracellular region of DLL4 (DLL4_IC) is required for receptor/ligand endocytosis, undergoes regulated intra-membrane proteolysis and, through its C-terminal PDZ binding motif, mediates the interaction of DLL4 with Dlg-1, a protein involved in the organization of cell-cell junctions. The sequence of DLL4_IC is very well conserved through evolution but does not encode any domain of known structure. Using a recombinant purified protein expressed from a codon-optimized synthetic gene, we demonstrate through various biophysical methods such as circular dichroism, size-exclusion chromatography, and NMR that DLL4_IC is globally disordered in solution, but can form inter-convertible local secondary structures in response to specific variations in the physico-chemical milieu, as well as in the presence of its target PDZ domain. Most of these conformational changes occur in the functionally relevant C-terminal segment. A computational study on the incidence and location of protein intrinsic disorder in 369 human receptors of the same transmembrane class of DLL4 provides evidence that disorder concentrates in the cytoplasmic tail of these proteins and represents a general phenomenon. In light of these findings, we propose that global disorder in the cytoplasmic tail, in concert with local pre-organization, may play a role in the function of DLL4 as well as in that of other single-pass transmembrane proteins.