Crystal structure of the human Scribble PDZ1 domain bound to the PDZ‐binding motif of APC

Abstract

Scribble (SCRIB) is an important adaptor protein that controls the establishment and maintenance of apico‐basal cell polarity. To better understand how SCRIB controls cell polarity signalling via its PDZ domains, we investigated human SCRIB interactions with adenomatous polyposis coli (APC). We show that SCRIB PDZ1, PDZ2 and PDZ3 are the major interactors with the APC PDZ‐binding motif (PBM), whereas SCRIB PDZ4 does not show detectable binding to APC. We then determined the crystal structure of SCRIB PDZ1 domain bound to the APC PBM. Our findings reveal a previously unreported pattern of interactions between the SCRIB PDZ domain region with the C‐terminal PDZ binding motif of APC, where SCRIB PDZ1 domain is the highest affinity site.

Structural basis of the human Scribble-Vangl2 association in health and disease

“…The overall mode of Vangl2 peptide engagement is conserved across all three PDZ domain complexes, with PDZ1, PDZ2 and PDZ3 engaging Vangl2 in a manner where the peptide is bound in an anti-parallel orientation compared to the second β-strand whilst being sandwiched by the α2 helix in the canonical binding pocket of the PDZ domains. A comparison of the PDZ1 and PDZ3 domain structures on their own with their Vangl2 complex counterparts revealed no significant movement of secondary structure elements upon Vangl2 binding, as previously observed in complexes of PDZ1 and PDZ3 with -PIX (Lim et al, 2017) and PDZ1 complexes with APC (How et al, 2019) and MCC (Caria et al, 2019 (Figure 2 B), key features of the PDZ1:Vangl2 complex are conserved, with V521 Vangl2 being located in a pocket formed by L872 PDZ2 , F874 PDZ2 , I876 PDZ2 , V932 PDZ2 and L935 PDZ2 , whilst the C-terminal carboxyl group forms hydrogen bonds with the main chain of L872 PDZ2 , G873 PDZ2 and F874 PDZ2 . Additional interactions are formed by S520 Vangl2 :S875 PDZ2 , T519 Vangl2 :H928 PDZ2 , as well as a salt bridge between E518 Vangl2 :S895 PDZ2 .…”

“…ITC measurements revealed that PDZ1E814G and PDZ3P1043L lost binding to both -Pix and APC in addition to Vangl2. In contrast, PDZ1Q808H and PDZ3R1044Q bound interactors with affinities comparable to wild-type PDZ (Lim et al, 2017) and APC (How et al, 2019), with PDZ1Q808H binding -Pix with 10.4 M, APC with 6.0 M and Vangl2 with 10.4 M, whereas PDZ3R1044Q bound -Pix with 14.1 M, APC with 27.4 M and Vangl2 with 20.9 M. PDZ1H793A showed only a significant decrease in binding to -Pix (62.77 M),…”

“…To confirm that the loss of binding of certain Scribble mutants was not due to an unfolding defect, we confirmed that all Scribble mutants were folded using circular dichroism spectroscopy ( Figure EV3). To determine whether or not the mutations in Scribble PDZ domain mutants were specific for Vangl2 or acted more broadly we examined the ability of PDZ1E814G, PDZ1Q808H, PDZ3P1043L and PDZ3R1044Q as well as our engineered mutants for other well-known Scribble interactors, such as -Pix (Lim et al, 2017) and APC (How et al, 2019). ITC measurements revealed that PDZ1E814G and PDZ3P1043L lost binding to both -Pix and APC in addition to Vangl2.…”

“…Each of the value was calculated from at least three independent experiments. Values denoted by a and b were taken from (Lim et al, 2017) and (How et al, 2019) WT lysates (left panel) and MCF10A cells stably expressing GFP-Vangl2 (right panel). Bound proteins were recovered with glutathione resin and revealed by western transfer using anti-GST, anti-β-PIX and anti-GFP antibodies.…”

The Scribble family in cancer: twentieth anniversary

“…Work from several labs has shown that Scribble directly interacts through its PDZ domains with proteins playing a role in different steps of cancer development and dissemination. Direct interactions have been reported with tumor suppressors mutated in cancer, Adenomatous polyposis coli (APC), a major component of Wnt signaling [39], the lipid phosphatase PTEN [40], MCC (Mutated Colorectal Cancer) [41], and with ZO-2, a junctional protein downregulated during cancer progression [42]. The Scribble PDZ domains also tightly bind to the PDZBM of the Guanine Exchange Factor (GEF) β-PIX/ARHGEF7 which promotes cancer cell migration [43,44].…”