Structural basis for Ccd1 auto-inhibition in the Wnt pathway through homomerization of the DIX domain

abstract: ...Wnt signaling plays an important role in governing cell fate decisions. Coiled-coil-DIX1 (Ccd1), Dishevelled (Dvl), and Axin are signaling proteins that regulate the canonical pathway by controlling the stability of a key signal transducer -catenin. These proteins contain the DIX domain with a ubiquitin-like fold, which mediates their interaction in the -catenin destruction complex through dynamic head-to-tail polymerization. Despite high sequence similarities, mammalian Ccd1 shows weaker stimulation of -catenin transcriptional activity compared with zebrafish (z) Ccd1 in cultured cells. Here, we show that the mouse (m) Ccd1 DIX domain displays weaker ability for homopolymerization than that of zCcd1. Furthermore, X-ray crystallographic analysis of mCcd1 and zCcd1 DIX domains revealed that mCcd1 was assembled into a double-helical filament by the insertion of the 1-2 loop into the head-to-tail interface, whereas zCcd1 formed a typical single-helical polymer similar to Dvl1 and Axin. The mutation in the contact interface of mCcd1 double-helical polymer changed the hydrodynamic properties of mCcd1 so that it acquired the ability to induce Wnt-specific transcriptional activity similar to zCcd1. These findings suggest a novel regulatory mechanism by which mCcd1 modulates Wnt signaling through auto-inhibition of dynamic head-to-tail homopolymerization.

... stimulation of -catenin transcriptional activity compared with zebrafish (z) Ccd1 in cultured cells. Here, we show that the mouse (m) Ccd1 DIX domain displays weaker ability for homopolymerization than that of zCcd1. Furthermore, X-ray crystallographic analysis of mCcd1 and zCcd1 DIX domains revealed that mCcd1 was assembled into a double-helical filament by the insertion of the 1-2 loop into the head-to-tail interface, whereas zCcd1 formed a typical single-helical polymer similar to Dvl1 and Axin. The mutation in the contact interface of mCcd1 double-helical polymer changed the hydrodynamic properties of mCcd1 so that it acquired the ability to induce Wnt-specific transcriptional activity similar to zCcd1. These findings suggest a novel regulatory mechanism by which mCcd1 modulates Wnt signaling through auto-inhibition of dynamic head-to-tail homopolymerization. issue-copyright-statement The Author(s) 2017 Introduction The Wnt signaling pathway plays key roles in cell fate determination during

... downstream signaling is unknown. Previously, we reported that mouse Ccd1 (mCcd1) showed low level of transcriptional activation compared to zebrafish Ccd1 (zCcd1), suggesting that mCcd1 signaling activity is controlled by homopolymerization mode distinct from those of other DIX-containing proteins 36 . Here, we report crystal structures of DIX domains in mCcd1 and zCcd1 resolved without additional mutagenesis or heavy atom modification. Interestingly, we found that the DIX domain of mCcd1 presented double-helical filaments formed by two head-to-tail helical polymers in crystal, whereas DIX domains of zCcd1 had a head-to-tail helical filament structure similar to that of the Hg-modified Axin DIX domain reported previously 32 . Point-directed mutagenesis of predicted structurally important residues followed by binding assays and transcriptional activity measurements revealed that loop 1-2 of the mCcd1 DIX domain inserted into head-to-tail interface prevented the formation of head-to-tail helical filaments and, consequently, inhibited mCcd1 transcriptional activity. These results disclose a novel regulatory mechanism of Wnt signaling in mammals through conformational auto-suppression of mCcd1, suggesting that the DIX domain functions as a switching hub in the canonical Wnt pathway via dynamic polymerization. Results mCcd1 DIX domain has a low affinity to dynamic homopolymerization Multiple amino acid sequence alignment of DIX domains of Ccd1, Dvl, and Axin showed that they had about 30% sequence

... homopolymers formed by mCcd1 and zCcd1 DIX domains. ( a ) Amino acid sequence alignment of DIX domains from Ccd1, Axin, and Dvl; secondary structural elements of mCcd1 are shown at the top. Conserved and semi-invariant residues (E=D, R=K=H, T=S, F=Y, V=L=I=M=C) are highlighted yellow and green, respectively, and acidic and basic residues are marked red and blue, respectively. Amino acids predicted to be involved in head-to-tail interactions are outlined by black boxes. The black and white circles indicate positions of M4 and M2 mutations, respectively. Residues unique in zCcd1 are shown in red boxes, and those involved in double-helical polymerization are marked by blue and brown circles. ( b d ) Hydrodynamic properties of mCcd1 and zCcd1 DIX domains. Gel filtration profiles of DIX domains of zCcd1 and mCcd1 ( b ). Gel filtration profiles of the MBP-fused mCcd1 DIX ( c ) and the MBP-fused zCcd1 DIX ( d ) at different loading concentrations. ( e , f ) Structures of mCcd1 double-helical ( e ) and zCcd1

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Gene: MCCD1 - mitochondrial coiled-coil domain 1 - (NCBI)

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