Refining a Jagged Edge

Abstract

In a recent Cell Reports article, Susan Lea, Penny Handford, and colleagues show that the N-terminal “MNNL” domain of the essential Notch ligand Jagged1 resembles a C2 domain, and relies on calcium binding to facilitate productive signal transduction (Chilakuri et al., 2013).

Notch signaling is a conserved cell-cell communication system that plays essential roles in development and tissue homeostasis in all metazoan organisms. In addition, dysregulated Notch signaling has been implicated in the pathogenesis of a number of human diseases, including cancer, underscoring its critical importance in human biology (Aster et al., 2008). Notch signals are initiated when ligands of the Delta or Jagged/Serrate families on a signal-sending cell bind to Notch receptors on signal-receiving cells. Ligand engagement triggers regulated proteolysis of Notch receptors, leading to release of the Notch intracellular domain (NICD) from the membrane and its eventual entry into the nucleus, where it regulates transcription of Notch target genes(see, for example, (Bray, 2006; Kopan and Ilagan, 2009) for comprehensive reviews).

Mammals have three Delta-like (Dll) ligands and two Jagged ligands, both homologous to Drosophila Serrate. These ligands (collectively referred to as “DSL” ligands) have a shared modular organization, which includes an N-terminal MNNL (Module at the N-terminus of Notch Ligands) domain, a cysteine-rich DSL (Delta, Serrate, Lag-2) module, and 6–16 EGF-like repeats. Previous studies have established that the MNNL and DSL elements of both Drosophila and mammalian ligands, along with at least EGF repeats 1–2, are required for binding to Notch receptors and for productive signaling(Andrawes et al., 2013; Parks et al., 2006; Shimizu et al., 1999). Similarly, loss-of-function missense mutations of Jagged1, which result in the developmental disorder Alagille’s syndrome, occur most frequently in the MNNL domain, the DSL domain, and the first two EGF repeats (Chillakuri et al., 2012).Despite accruing evidence that the MNNL domain plays an important role in ligand-dependent Notch signaling, however, how it participates in Notch activation has remained elusive.

In a recent Cell Reports article, Lea, Handford, and colleagues now address the structure and function of the MNNL domain of Jagged1 through a combination of structural, biochemical, and cell-based assays (Chilakuri, 2013). First, they determined the X-ray crystal structure of a Jagged1 fragment that encompasses the MNNL, DSL, and first three EGF-like modules (denoted JAG1_N-EGF3). The structure bears some resemblance to the Olympic torch, with the MNNL domain as the flame and the DSL-EGF3 region (solved previously; (Cordle et al., 2008)) acting as the torch handle (Figure 1A). The keynew structural finding, entirely unexpected based on sequence alignment or use of other informatics tools, is that the MNNL domain bears striking structural similarity to C2 domains. Canonical C2 domains, such as those found in protein kinase C (PKCα: pdb ID code: 1DSY) or Munc13 (pdb ID code: 3KWU) are beta-sandwich structures that typically contain one or two calcium binding sites derived from either the β2-β3 and β6-β7 loops (PKCα) or the β1-β2 and β5-β6 loops (Munc13) at one end of the domain (Figure 1B). The calcium-binding loops in these proteins often act as switches for recruiting the C2 domains to the plasma membrane by creating binding sites for phospholipids such as phosphatidylserine upon calcium loading(Lemmon, 2008).

Figure 1.

Structure of the Jagged N-terminal region and comparison with C2 domains from PKCα and MUNC13. A. Transparent surface over a ribbon representation of the JAG1_N-EGF3 fragment. The MNNL domain is orange, and the DSL-EGF3 stem region is colored blue. The bound calcium ion is gray. B. Structural comparison of the Jagged1 MNNL domain (top, gold) with the C2 domains of Munc13 (middle, cyan) and Protein kinase Cα (bottom, green). The view shown is rotated clockwise by approximately 90 degrees from that in panel A. The backbone is rendered with a ribbon diagram, side chains participating in calcium coordination are illustrated as sticks, and calcium ions are rendered as gray spheres. The carbon backbone of the phosphatidylserine bound to the PKCα C2 domain is yellow, and heteroatoms are in CPK colors.

What about the calcium-binding properties of the Jagged1 MNNL domain, and the implications of calcium loading for ligand-induced Notch signaling? A second X-ray crystal structure of the JAG1_N-EGF3fragment solved in the presence of calcium reveals a bound Ca⁺⁺ ion in a binding site formed by acidic residues derived from the β1-β2 and β5-β6 loops, as predicted based on alignment with Munc13.The authors then proceed to show that JAG1_N-EGF3, as well as homologous regions of human Delta-like 1 and Drosophila Serrate, bind liposomes in a calcium dependent fashion, and that deletion of the MNNL domain reduces liposome binding to background levels. Moreover, mutations that alter the calcium binding residues do not appear to affect the affinity of these proteins for Notch receptors, but the mutated proteins are nevertheless deficient at inducing Notch signal transduction in a reporter-based assay for activity.

Together these findings appear to support the proposal that calcium loading of the C2 domain facilitates a lipid-binding step that is important for Notch ligand function, but several questions still remain. Does the MNNL domain of Jagged1 directly engage the membrane of the signal-receiving cell that expresses the Notch receptor? Or alternatively, could phospholipid binding by Jagged1 be a proxy interaction for the true calcium-dependent partner of the MNNL domain, perhaps another protein or even a complementary trigger site on Notch itself? The Cell Reports article from Lea, Handford and colleagues not only gives new insights into the structure and biochemical properties of Notch ligands, but also opens the door to new lines of experimentation in the Notch field that should provide definitive answers to these and other questions in the near future.