Insights into Chibby\u27s structural elements and their interplay in Wnt signaling protein-protein interactions

The Wnt/b-catenin signaling pathway is critical to embryonic development and adult tissue homeostasis. Mutations to Wnt signaling components can cause dysregulation of the pathway, leading to various human diseases such as cancer. The partially disordered protein Chibby (Cby) is a conserved nuclear protein that acts as an antagonist in the Wnt/b-catenin signaling pathway. Cby’s antagonism is accomplished via two mechanisms. First, by competing with the Tcf/Lef family of transcription factors, Cby abrogates the b-catenin-mediated transcription of Wnt signaling genes. Moreover, upon phosphorylation on serine 20 by the kinase Akt, Cby forms a complex with the protein 14-3-3 to facilitate the nuclear export of b-catenin. Structurally, Cby is composed of an unstructured N-terminal half, while its C-terminal half harbours a coiled-coil domain. Cby’s N-terminal half comprises a 14-3-3 binding motif, while its C-terminal half mediates the interaction with b-catenin, as well as TC-1, an antagonist of Cby. In this thesis, the molecular details of Cby’s structural elements and its interactions with the Wnt signaling components 14-3-3, b-catenin and TC-1 were investigated. The Cby/14-3-3 interaction was studied by using a combined approach of nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry (ITC) and X-ray crystallography. While the solved crystal structure revealed a canonical binding mode, NMR spectroscopy and ITC revealed that residues flanking Cby’s 14-3-3 binding motif are involved in the interaction. Next, hydrogen-deuterium exchange mass spectrometry revealed that in addition to Cby’s disordered N-terminus, Cby contains a disordered C-terminal extension. ITC and NMR experiments demonstrate that the disordered N-terminus negatively regulates target binding between TC-1 and Cby’s coiled-coil domain. Lastly, mutagenesis studies suggest that Cby’s coiled-coil domain utilizes differing binding modes when interacting with b-catenin and TC-1, with Cby binding as a monomer to b-catenin and as a dimer to TC-1. In conclusion, this thesis demonstrates how Cby’s structural elements collectively mediate protein-protein interactions in the Wnt signaling pathway

Molecular effects of cancer-associated somatic mutations on the structural and target recognition properties of Keap1.

Kelch-like ECH-associated protein 1 (Keap1) plays an important regulatory role in the nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent oxidative stress response pathway. It functions as a repressor of Nrf2, a key transcription factor that initiates the expression of cytoprotective enzymes during oxidative stress to protect cells from damage caused by reactive oxygen species. Recent studies show that mutations of Keap1 can lead to aberrant activation of the antioxidant pathway, which is associated with different types of cancers. To gain a mechanistic understanding of the links between Keap1 mutations and cancer pathogenesis, we have investigated the molecular effects of a series of mutations (G333C, G350S, G364C, G379D, R413L, R415G, A427V, G430C and G476R) on the structural and target recognition properties of Keap1 by using nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) and isothermal titration calorimetry (ITC). Depending on their locations in the protein, these mutations are found to exert differential effects on the protein stability and target binding. Together with the proposed hinge-and-latch mechanism of Nrf2-Keap1 binding in the literature, our results provide important insight into the molecular affect of different somatic mutations on Keap1\u27s function as an Nrf2 repressor

Structure of the DOCK2-ELMO1 complex provides insights into regulation of the auto-inhibited state.

Funder: Gouvernement du Canada | Instituts de Recherche en Santé du Canada | CIHR Skin Research Training Centre (Skin Research Training Centre); doi: https://doi.org/10.13039/501100007202Funder: Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada (NSERC Canadian Network for Research and Innovation in Machining Technology); doi: https://doi.org/10.13039/501100002790DOCK (dedicator of cytokinesis) proteins are multidomain guanine nucleotide exchange factors (GEFs) for RHO GTPases that regulate intracellular actin dynamics. DOCK proteins share catalytic (DOCKDHR2) and membrane-associated (DOCKDHR1) domains. The structurally-related DOCK1 and DOCK2 GEFs are specific for RAC, and require ELMO (engulfment and cell motility) proteins for function. The N-terminal RAS-binding domain (RBD) of ELMO (ELMORBD) interacts with RHOG to modulate DOCK1/2 activity. Here, we determine the cryo-EM structures of DOCK2-ELMO1 alone, and as a ternary complex with RAC1, together with the crystal structure of a RHOG-ELMO2RBD complex. The binary DOCK2-ELMO1 complex adopts a closed, auto-inhibited conformation. Relief of auto-inhibition to an active, open state, due to a conformational change of the ELMO1 subunit, exposes binding sites for RAC1 on DOCK2DHR2, and RHOG and BAI GPCRs on ELMO1. Our structure explains how up-stream effectors, including DOCK2 and ELMO1 phosphorylation, destabilise the auto-inhibited state to promote an active GEF

NMR titration experiments of 14-3-3ζΔC12 with Cby peptides.

<p>(A) ¹H-¹⁵N TROSY-HSQC spectra of 14-3-3ζΔC12 alone (black) and with 3 molar equivalents of the Cby 7-mer (red) and Cby 18-mer (blue). (B) ¹H-¹⁵N TROSY-HSQC spectra of 14-3-3ζΔC12 alone (black) and with 3 molar equivalents of the WT Cby 18-mer (blue) and Cby S22P 18-mer (blue).</p

Crystallographic data collection and refinement statistics.

<p>Values in parentheses refer to the highest resolution shell.</p><p>Crystallographic data collection and refinement statistics.</p

The mapping of chemical shifts on the crystal structure of the 14-3-3ζ /Cby complex.

<p>Due to the crowding of some peaks, the chemical shifts of some residues could not be confidently traced and were excluded from the analysis. (A) (Above) A monomer of the 14-3-3ζ /Cby crystal structure interface based on Cby residues (¹⁸SApSLSNLH²⁵). Residues R56, R127 and Y128 which contact the Cby phosphate group are coloured red, residues interfacing the peptide are coloured cyan, and 14-3-3ζ residues coloured magenta are found along the 14-3-3 dimer interface. (Below) The 14-3-3ζ dimer bound to Cby. (B) (Cby 7-mer), (C) (Cby 18-mer) and (D) (Cby S22P 18-mer). Residues with traceable assigned resonances are coloured on a blue—white—yellow gradient (0 ppm to 0.1 ppm) based on their combined chemical shift [Δω = ((Δδ¹H)² +(0.2*Δδ¹⁵N)²)^1/2] at a 3:1 peptide:protein ratio. Residues coloured in orange represent peaks that broadened out to disappearance upon addition of peptide. Residues R56, R127 and Y128 are coloured pink.</p

The 14-3-3ζ, Cby, β-catenin tripartite complex.

<p>Model of dimeric 14-3-3ζ bound to two molecules of full-length Cby. The models of two full-length Cby proteins have disordered residues 30–73 of the N-terminus and 100–126 of the C-terminus presented in a highly extended fashion, and are shown forming a coiled-coil from residues 73–100. β-catenin (PDB: 2Z6H) is shown with its Cby binding-site (helix C) in red which then binds along the C-terminus (64–126) of Cby.</p

Structural comparison of 14-3-3ζ-bound Cby with other 14-3-3 binding motifs comprising various +2 residues.

<p>Residues K49 and R56 are coloured yellow on the white surface representation of 14-3-3ζ. Structural and sequence alignment of Cby (green sticks) with the binding-motifs of (A). Raf1 (pink sticks, PDB: 3CU8), (B) PKCε (orange sticks, PDB: 2WH0), (C) Histone H3 (white sticks, PDB: 2C1N), (D) β2 integrin (blue sticks, PDB: 2V7D) and (E) α 4 integrin (purple sticks. PDB: 4HKC). The Cα RMSD values were computed by subtracting a Cα distance matrix between the -2 and +1 residues of Cby and each peptide as well as the -2 and most C-terminal residue in the respective alignments.</p