Autoinhibition of UNC5b Revealed by the Cytoplasmic Domain Structure of the Receptor

Abstract

The cytoplasmic domains of UNC5 are responsible for its netrin-mediated signaling events in axonal migrations, blood vessel patterning, and apoptosis, although the molecular mechanisms governing these processes are unknown. To provide a foundation for the elucidation of the UNC5-mediated signaling mechanism, we determined the crystal structure of the cytoplasmic portion of UNC5b. We found that it contains three distinctly folded domains, namely ZU5, UPA, and death domain (DD). These three domains form a structural supramodule, with ZU5 binding to both UPA and DD, thereby locking the ZU5-UPA-DD supramodule in a closed conformation and suppressing its biological activities. Release of the closed conformation of the ZU5-UPA-DD supramodule leads to the activation of the receptor in the promotion of apoptosis and blood vessel patterning. Finally, we provide evidence showing that the supramodular nature of UNC5 ZU5-UPA-DD is likely to be shared by the ankyrin and PIDD families of scaffold proteins.

Dependence receptors: a new paradigm in cell signaling and cancer therapy

“…On this basis, and according to their observations, Llambi and colleagues proposed that, in the presence of netrin-1, DAPK is in an inactive autophosphorylated state, and it interacts with UNC5H2 through their non-'death domain'-interacting regions, whereas, in the absence of netrin-1, DAPK interacts with the death domain of UNC5H2, which allows DAPK activation ( Figure 5). This hypothesis strongly fits with the more recent study by Wang et al (2009), arguing that UNC5H2 can adopt a closed conformation, preventing association of the death domain with other proteins. Downstream effectors of DAPK in UNC5H2-mediated cell death remain to be identified, but DAPK is already known to trigger cell death through p53-dependent and -independent mechanisms.…”

“…In this conformation, ZU5 and death domains bind to each other and are thus unable to induce cell death. Netrin-1 is unable to prevent apoptosis induced by the UNC5H2 mutant that has a constitutive open conformation, which leads the authors to suggest that netrin-1 somehow stabilizes the autoinhibited conformation of UNC5H2 (Wang et al, 2009).…”

The death domain-containing protein Unc5CL is a novel MyD88-independent activator of the pro-inflammatory IRAK signaling cascade

“…17 Interestingly, the resolution of the X-ray crystal structure of the rat Unc5B intracellular domain revealed a tripartite domain organization comprising the previously described ZU5 and DD, as well as a novel central UPA domain. 16 To evaluate whether Unc5CL also contains such a ZU5-UPA-DD supramodule we used Jpred3 to predict the secondary structure of Unc5CL and Triangle: specific site of cleavage; black shading: identical amino acids; gray shading: similar amino acids. (c) HEK293T cells were transfected with the indicated C-terminally FLAG-tagged Unc5CL point mutants.…”

“…14,15 A functionally heterogeneous subgroup of these proteins is characterized by the presence of a tripartite domain module, termed the ZU5-UPA-DD supramodule, which in addition to a DD, contain a ZU5 (domain present in ZO-1 and Unc5) and a UPA (domain conserved in Unc5, PIDD and Ankyrin) domain. 16 In mammals, this family comprises PIDD (p53-induced protein with a DD), Ankyrins1-3, the transmembrane receptors Unc5A-D and the poorly characterized protein Unc5CL (Figure 1a). Based on the resolution of the crystal structure of the intracellular part of Unc5B, Wang et al 16 proposed a conserved activation mechanism for these molecules, in which the ZU5 domain sequesters both the UPA and DD, keeping them in an auto-repressed state.…”

“…16 In mammals, this family comprises PIDD (p53-induced protein with a DD), Ankyrins1-3, the transmembrane receptors Unc5A-D and the poorly characterized protein Unc5CL (Figure 1a). Based on the resolution of the crystal structure of the intracellular part of Unc5B, Wang et al 16 proposed a conserved activation mechanism for these molecules, in which the ZU5 domain sequesters both the UPA and DD, keeping them in an auto-repressed state.…”