Structural analysis of phosphorylation-associated interactions of human MCC to Scribble PDZ domains

Scribble is a crucial adaptor protein that plays a pivotal role during establishment and control of cell polarity, impacting many physiological processes ranging from cell migration to immunity and organization of tissue architecture. Scribble harbors a leucine-rich repeat domain and four PDZ domains, which mediate most of Scribble’s interactions with other proteins. It has become increasingly clear that posttranslational modifications substantially impact Scribble-ligand interactions, with phosphorylation being a major modulator of binding to Scribble. To better understand how Scribble PDZ domains direct cell polarity signalling and how phosphorylation impacts this process, we investigated human Scribble interactions with MCC (mutated in colorectal cancer). We systematically evaluated the ability of all four individual Scribble PDZ domains to bind the PDZ-binding motif (PBM) of MCC as well as MCC phosphorylated at the -1 Ser position. We show that Scribble PDZ1 and PDZ3 are the major interactors with MCC, and that modifications to Ser at the -1 position in the MCC PBM only has a modest effect on binding to Scribble PDZ domains. We then examined the structural basis for these observations by determining the crystal structures of Scribble PDZ1 domain bound to both the unphosphorylated MCC PBM as well as phosphorylated MCC. Our structures indicated that phospho-Ser at the -1 position in MCC is not involved in major contacts with Scribble PDZ1, and in conjunction with our affinity measurements suggest that the impact of phosphorylation at the -1 position of MCC extends beyond a simple modulation of the affinity for Scribble PDZ domains.


INTRODUCTION
Cell polarity, which manifests itself as the asymmetric distribution of cellular constituents as well as proteins, lipids and carbohydrates into distinct cellular domains (1), is a critical property of eukaryotic cells and pivotal for correct tissue architecture and tissue development. The particular distribution of cellular constituents leads to the establishment of apical-basal cell polarity in epithelial cells, and impacts numerous critical cellular signaling pathways including those involved in apoptosis, vesicle trafficking, cell proliferation and migration (2). Furthermore, loss of cell polarity is recognized as an important hallmark of cancer development (3), underscoring the importance of correct cell polarity for healthy tissues. Apico-basal polarity is controlled by the interplay of three multi-protein complexes, Par, Crumbs and Scribble, with epithelial cell polarity being orchestrated by the antagonistic interaction between PAR and Crumbs with the Scribble complex. In mammals, the Scribble complex comprises Scribble (SCRIB), one of 4 Dlg (Discs Large) homologs (DLG1-4) and 2 Lgl (Lethal Giant Larvae) homologs (LLGL1, LLGL2) which are highly conserved from the vinegar fly to humans (4). Scribble was identified in Drosophila melanogaster as a tumour suppressor where loss of Scribble resulted in disrupted epithelial tissue organization accompanied by aberrant growth in the imaginal discs of the larvae (5). The ability to suppress tumors was shown to be conserved across species, with Scribble knock-out promoting tumor initiation, and when coupled with oncogenic drivers including RAS, tumor progression in diverse epithelial tissues including mammary, prostate, skin and the lung (6)(7)(8)(9)(10).
Scribble is a large multi-domain scaffold protein comprising 16 Leucine Rich Repeats and 4 PSD-95/Disc-large/ZO-1 (PDZ) domains, and belongs to the LAP family of proteins. These domains enable Scribble to interact with a diverse set of interactors that play a role in a range of discrete signaling pathways (4). Whilst the majority of interactions are modulated by the four PDZ domains, the Scribble LRR domain also engages a specific subset of interactions, such as with Lgl2, during the regulation of cell polarity (11).
Binding to Scribble via its PDZ domains is typically mediated via C-terminally located PDZ-binding motifs (PBMs) on specific interactors. Although these sequences are specific, several studies have shown that whilst PBMs on Scribble interactors selectively engage Scribble, the Scribble PDZ domains appear to harbor overlapping preferences for certain ligands, with each PDZ domain capable of engaging multiple binding partners. Scribble PDZ domains are classified as Class I PDZ domains, which recognizes a consensus X-T/S-X-ØCOOH motif (where X can be any amino acid residue, and Ø is a hydrophobic residue) in the PBM of interactors. In addition to amino acid variations, post-translational modification via phosphorylation has also been shown to impact ligand binding to PDZ domains. Binding of Kir2.3 (12) and stargazin (13) to DLG4 (PSD-95) is regulated by phosphorylation, with phosphorylation of a Thr at the -2 position in stargazin abrogating binding to DLG4. Similarly, phosphorylation of a Ser at -2 prevents Kir2.3 binding to DLG4. More recently phosphorylation of PBMs was examined using proteomics approaches, which suggested that phosphorylation of PBMs occurs frequently and can have diverse impacts on PDZ domain binding (14). A comprehensive analysis of the effect of phosphorylation in the PBM of R58 in position -2, -3, -5 and -6 on the binding to the SNX27 PDZ domain (15) revealed that phosphorylation of the -2 position blocks the interaction, whereas phosphorylation at the -3 and -5 position significantly increased affinity.
MCC (mutated in colorectal cancer) has been shown to be a Scribble interactor (16). This complex appears to be highly conserved evolutionarily as Drosophila Scrib and MCC can interact physically (16) and in zebrafish loss of Scrib and MCC interact genetically to regulate convergent extension movements during gastrulation (17). MCC is a large multi-PDZ domain protein found in epithelial cells that has been shown to participate in several key cellular signaling pathways including canonical and non-canonical Wnt signaling, NFKB signaling as well as cell cycle control (17)(18)(19)(20). More recently, it was shown that MCC harbors a C-terminal PBM that is able to engage human Scribble PDZ1 and 3 domains to impact epithelial cell polarity (16). Interestingly, the MCC PBM has been shown to be a target for phosphorylation in position -1 which features a Ser residue, with phosphorylation impacting the formation of lamellipodia in colon epithelial cells (21). Apart from MCC only Syndecan1 has been described to feature phosphorylation a the -1 position of their PBM (22), which promoted binding to the Tiam1 PDZ domain by improving the affinity from 51.

Isolated human Scribble PDZ domains specifically interact with the b-PIX PBM
Human Scribble has previously been shown to directly interact with the MCC C-terminal PDZ binding motif (PBM) via its PDZ1 and 3 domains using pull-down and biochemical assays. To  Table 1).
Examination of the thermodynamic binding parameters of MCC binding to Scribble PDZ3 indicates that the modestly tighter binding of phosphorylated MCC (pMCC) to Scribble PDZ3 compared to the unphosphorylated MCC peptide is largely driven by a more favorable -TDS or entropic term, indicative of more disorder upon pMCC binding ( Figure 3, Table 1).

The crystal structures of SCRIB PDZ1:MCC peptides
To understand the structural implications of Ser phosphorylation at the -1 site of the MCC PBM we next determined the crystal structure of PDZ1 bound to the MCC 8-mer peptide ( Figure 4, 5, Table 2). As previously shown (24) PDZ1 adopts a compact globular fold comprising six β-strands and two α-helices that form a β-sandwich structure. The MCC peptide is bound in the canonical being resolved. A comparison of the PDZ1:MCC complex with PDZ1-B:pMCC-C reveals that R762 PDZ1 also forms an ionic bond with the phosphate group on pS828 pMCC , however in this instance the bond is formed with the actual pMCC chain bound in the PDZ1 domain binding groove. In addition, hydrogen bonds between S761 PDZ1 and E826 pMCC as well as S748 PDZ1 and N825 pMCC and G747 PDZ1 and H823 pMCC are found. Interestingly, the T824 MCC :T749 PDZ1 hydrogen bond seen in the PDZ1:MCC complex is absent in the PDZ1:pMCC-C, and instead T749 PDZ1 engages P822 pMCC-D .
Lastly, the C-terminal pMCC residue L829 only makes two contacts via its carboxyl group with

DISCUSSION
Scribble is an important regulator of apico-basal cell polarity, and enables the integration of a diverse set of signalling pathways that all converge on Scribble via interactions using its four PDZ domains. An added layer of complexity to the interactions of Scribble with its interactors via PDZ domains is the possibility of post-translational modifications that alter the affinity with which Scribble binds to its partners. Indeed, several Scribble-ligand interactions have now been shown to be affected by post-translational modifications, suggesting that phosphorylation may be a major regulatory mechanism for Scribble-ligand interactions (14,21). Intriguingly, phosphorylation does not always impact Scribble PDZ domain binding. Phosphorylation of APC seems to have little effect on Scribble binding, whereas phosphorylation of RPS6KA2 leads to a 4-fold increase in affinity for Scribble PDZ1. In contrast, phosphorylation of MAPK12 completely abolishes Scribble PDZ1 interaction (14).
It has previously been shown that phosphorylation of MCC impacts binding to Scribble PDZ domains, with phospho-MCC showing a modest 2-fold increase in affinity for Scribble PDZ1 (14). Similarly, phospho-MCC has been predicted to show tighter binding to Scribble PDZ3 (21).
However, NMR chemical shift analysis revealed no substantial differences for Scribble PDZ1 binding to phosphorylated and unphosphorylated MCC that could provide a structural rational for the reported increased affinity (23). Although MCC is able to bind to both Scribble PDZ1 and 3 domains, the interaction with PDZ3 is substantially tighter. Interestingly, PDZ3 has been shown to be the primary binding site for MCC (16), with phosphorylation of S828 at the -1 position predicted to lead to tighter binding compared to unphosphorylated MCC (21) based on functional data where enzymatically dephosphorylated MCC showed reduced levels of immunoprecipitation with full length Scribble.
Unexpectedly, our data suggest that phosphorylation of MCC -1 S828 does not significantly alter its affinity for Scribble PDZ3. Pangon et al. (21) previously observed that there is no significant difference in immunoprecipitation levels between unphosphorylated MCC and phosphorylated MCC with Scribble, whereas an MCC S828A mutant displayed reduced Scribble binding. These findings were interpreted as evidence that MCC is usually phosphorylated. Our data suggest that MCC may not necessarily be phosphorylated to account for the lack of significant differences in immuno-precipitation levels observed, and may be indicative of an effect that is not directly linked to Scribble binding. This is in contrast to MCC binding to Dlg, where phosphorylation of MCC leads to a decrease of affinity from 2.7 to 95 µM for Dlg PDZ2 (14).
Examination of the thermodynamics of MCC and pMCC binding to PDZ3 reveal that the tighter binding of pMCC to PDZ3 is due to more favorable -TDS or entropy terms rather than enthalpy terms, which remained the same. We speculate that few or no major additional hydrogen bonds are formed with the phosphate group on Ser -1, however this would have to be confirmed by direct structure determination of a PDZ3:pMCC complex.
Compared to other previously established affinities of Scribble PDZ1 domain for interactors, MCC is not a particularly high affinity interactor. Gukh engages Drosophila Scribble PDZ1 with 660 nM affinity (26), and b-PIX binds human Scribble PDZ1 with 2 µM affinity (24,27). MCC only binds to Scribble PDZ1 with 7.7 µM affinity, and examination of the different molecular contacts between MCC and Scribble PDZ1 indicate that the lower affinity compared to b-PIX may be due to the lack of a hydrophobic residue engaging the PDZ1 b2-3 loop, as is achieved by b-PIX via b-PIX W641 (24) or Trp at position -4 in a high affinity nanomolar complex of a synthetic peptide with Erbin (28) (see Figure 5D). An alternative configuration that enables high affinity interactions is observed in the Scribble PDZ1:Gukh complex, where F1784 in Gukh contacts H796 on the opposite side of the ligand binding groove (26).
In summary, we show that Scribble PDZ3 domain binds MCC with a ~2-fold higher affinity compared to Scribble PDZ1 domain. Furthermore, phosphorylation of MCC at the -1 position of the PBM only has a modest impact on binding to Scribble PDZ1 and PDZ3 domains, suggesting that the impact of phosphorylation of the MCC PBM at the -1 position extends beyond a simple change in binding affinity to Scribble PDZ domains and that could be dependent on a Scribble PDZ interdomain interaction to favour or block MCC binding. Another possibility is that -1 phosphorylation of the MCC PBM primarily affects an interactor that indirectly affects Scribble such as Dlg, which has been shown to display substantial modulation of affinity for MCC depending on the phosphorylation status of the MCC PBM.
In summary, our findings provide a clear structural basis for Scribble PDZ1:MCC interactions, and will enable more detailed structure-guided investigations to understand the precise effect of MCC phosphorylation on the control of cell polarity and directed migration.

Protein crystallisation, data collection and refinement
Complexes of PDZ1 with MCC and pMCC peptides were reconstituted by mixing protein and peptide at a 1:2 molar ratio. The dilute protein complexes were then concentrated to 15 and 23 mg/ml respectively, using a 3-kDa molecular mass cut-off centrifugal concentrator (Millipore), flash-cooled, and stored under liquid nitrogen. Crystallization trials were carried out using 96-well All diffraction data were collected on the MX2 beamline at the Australian Synchrotron using an Eiger 16M detector with an oscillation range of 0.1° per frame using a wavelength of 0.9537 Å. PDZ1:MCC diffraction data was integrated with Xia2 (32) and scaled using AIMLESS (33) while PDZ1-pMCC data was integrated with XDSme (34) to detector resolution. PDZ1-pMCC diffraction was anisotropic with resolution limits of 2.8 Å along the a* and b* orientation and 2.1 Å on the c* orientation. Therefore, the data was elliptically truncated and corrected using the Staraniso server (http://staraniso.global phasing.org). The solutions produced by Phaser was manually rebuilt over multiple cycles using Coot (36) and refined using PHENIX (37). Data collection and refinement statistics details are summarized in Table 2. MolProbity scores were obtained from the MolProbity web server (38). Coordinate files have been deposited in the Protein Data Bank under the accession codes 6MTU and 6MTV. All images were generated using the PyMOL Molecular Graphics System, Version 1.8 Schrödinger, LLC. All software was accessed using the SBGrid suite (39). All raw diffraction images were deposited on the SBGrid Data Bank (40) using their PDB accession code 6MTU and 6MTV.      shown as a cartoon with unphosphorylated MCC peptide (green) represented as sticks.

TABLES
(B,C) PDZ1 (grey and yellow) is shown as a cartoon with phosphorylated MCC peptide (cyan or coral) represented as sticks. Both copies of the complexes formed by PDZ1 chain A with pMCC chainD as well as PDZ1 chain B and pMCC chain C are shown.