'Scuola Normale Superiore - Edizioni della Normale'
Abstract
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