Our results, combined with the studies of (Bourhis et al., 2010
), indicate that the two conserved regions of Dkk1 can inhibit all Wnts from binding to LRP5/6. LRP5/6 repeat 3, which binds to Dkk1_C, is needed for Dkk1 binding and Wnt1 antagonism (Mao et al., 2001a
; Zhang et al., 2004
), yet Wnt1 and many other Wnt variants appear to bind the LRP5/6(1-2) region (Gong et al., 2010
). Moreover, Dkk1_N by itself does not appear to inhibit signaling (Brott and Sokol, 2002
; Mao and Niehrs, 2003
). Since Dkk1_N binds to the LRP6(1-2) region, the interaction of Dkk1_C with repeat 3 likely provides a high-affinity anchor that makes Dkk1_N an effective inhibitor of Wnt binding at physiological concentrations, consistent with the 21x stronger binding of Dkk1 to LRP6(1-4) compared to LRP6(1-2) (Bourhis et al., 2010
). Signaling by Wnts 1 and 8, which appear to bind LRP6(1-2) (Gong et al., 2010
), can be inhibited by Dkk1_C although not as strongly as by full-length Dkk1 (Brott and Sokol, 2002
; Mao and Niehrs, 2003
). We suggest that interaction with Kremen receptors and receptor internalization, or inhibition of receptor dimerization, mediate these effects (Binnerts et al., 2009
; Gong et al., 2010
The Dkk_N region is not as strongly conserved as Dkk_C (e.g.,
40% identity between human Dkk1_N and Dkk2_N, versus 67% for Dkk1_C and Dkk2_C), and studies with deletion and chimeric constructs suggest that the differences between Dkk1 and 2 reside in Dkk_N (Brott and Sokol, 2002
). The distinct biological effects of Dkk1, 2, and 4 (Brott and Sokol, 2002
; Krupnik et al., 1999
) may thus arise from differences in their interactions with the LRP5/6(1-2) region.
Dkks likely inhibit Wnt signaling by directly competing with Wnts for binding sites on LRP5/6. However, HBM mutations in LRP5 diminish Dkk1, but not Wnt, binding to LRP5 (Ai et al., 2005
; Balemans et al., 2007
), indicating that the Wnt binding site on LRP5/6(1-2) is distinct from this portion of the repeat 1 surface (Figure S5
) required for Dkk1_N binding. The other portion of the repeat 1 amphitheater might contribute to the Wnt interface, as well as the repeat 2 propeller. LRP5 missense mutations R494Q, R570W and V667M that cause OPPG and show diminished Wnt signaling (Gong et al., 2001
) lie dispersed on the side of the repeat 2 βpropeller barrel. A naturally occurring mutation, R611C in LRP6 repeat 2 impairs Wnt3a-mediated signaling (Mani et al., 2007
). This position is equivalent to Val913 and Arg1227 of repeats 3 and 4, which mediate interactions between the β-propeller and EGF-like domains within a repeat and likely stabilize their relative positions. We speculate that these mutations may directly prevent Wnt interactions with repeat 2, or they might alter the receptor conformation so as to make the Wnt binding site inaccessible or prevent receptor clustering.
Deletion of the entire extracellular region of LRP6 results in a constitutively active receptor, indicating that unliganded LRP5/6 is autoinhibited (Liu et al., 2003
; Mao et al., 2001a
; Mao et al., 2001b
). Thus, Dkks may stabilize an autoinhibited conformation, whereas a Wnt might stabilize a distinct structure. The conformation of LRP6 bound to mAb135 should be “active” since the antibody enhances Wnt3a signaling (Binnerts et al., 2009
). The LRP6(1-4) model derived from the Fab135 SAXS data appears compatible with the LRP6(1-4)–Dkk1 envelope, so potential conformational differences would appear to be small. Note, however, that the uncertainties of these low-resolution (~40 Å) reconstructions do not allow us to conclude that the receptor conformation the same in both cases.
Wnt signaling appears to depend upon the ability of a particular Wnt to bind simultaneously to LRP5/6 and a Fzd (He et al., 2004
; MacDonald et al., 2009
). The orientation and distance of the LRP5/6(1-4) region with respect to the membrane cannot be assessed without knowledge of the intervening LDLR-A repeats, but the overall conformation seen in the SAXS model of the Fab complex suggests that repeats 1, 2 and 3 could lie at roughly the same distance from the membrane (). Thus, a Wnt bound to either portion of the LRP5/6 ectodomain could access the Fzd CRD. The overall conformation of the LRP6 ectodomain also suggests that it may be able to engage two Wnt/Fzd complexes simultaneously (Bourhis et al., 2010
), but further biophysical and functional studies will be needed to assess this possibility.