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

Structural Characterization of Minor Ampullate Spidroin Domains and Their Distinct Roles in Fibroin Solubility and Fiber Formation

10.1371/journal.pone.0056142PLoS ONE82

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

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