1. Volume 25, Issue 11, Pages 1645-1656.e5 (November 2017)
Structural Characterization of Whirlin Reveals an Unexpected and Dynamic Supramodule Conformation of Its PDZ Tandem 
Florent Delhommel, Florence Cordier, Benjamin Bardiaux, Guillaume Bouvier, Baptiste Colcombet-Cazenave, Sébastien Brier, Bertrand Raynal, Sylvie Nouaille, Amel Bahloul, Julia Chamot-Rooke, Michael Nilges, Christine Petit, Nicolas Wolff 
Structure 
Volume 25, Issue 11, Pages e5 (November 2017)
DOI: /j.str
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2. Structure 2017 25, 1645-1656.e5DOI: (10.1016/j.str.2017.08.013)
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3. Figure 1 Whirlin PDZ1-2 Tandem Dynamic in Solution
(A) Top: Domain organization of whirlin from Mus musculus with the three main isoforms. The red extremities of the short isoforms indicate sequence differences from alternative splicing. Bottom: The various constructs used in this study. The wild-type tandem of PDZ is named PDZ1-2 (or PDZ1-2WT), the “structural mutants” were designed for fluorescence, SAXS, and NMR chemical shift perturbation experiments and the “PRE mutants” for NMR paramagnetic relaxation enhancement experiments (mutation positions indicated by stars).
(B) Alignment of whirlin PDZ1-Hp1 (orange) and PDZ2-Hp2 (green) with their secondary structures.
(C) Averaged 15N longitudinal and transverse relaxation times (25°C and 600 MHz) and correlation times of PDZ1-Hp1, PDZ2-Hp2, and the tandem PDZ1-2.
(D) Tryptophan emission fluorescence spectra of PDZ1-Hp1, PDZ2-Hp2, and PDZ1-2.
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4. Figure 2
Effect of Mutation in the C-Terminal Hairpin Extensions on the Overall Conformation of PDZ1-2
(A) Substitutions performed on each PDZ hairpin (black for PDZ1-2WT, light blue for PDZ1-2IP225GG, light red for PDZ1-2W237G, dark red for PDZ1-2LP363GG, and dark blue for PDZ1-2W375G; the mutant color coding is conserved throughout the figures). The gray boxes delineate the expected strands from TalosN analysis and the homologous harmonin model (PDB: 3K1R).
(B) Scattering curves of PDZ1-2WT and PDZ1-2 mutants (top panel) and the residual between the scattering curves of PDZ1-2WT and PDZ1-2 mutants (bottom panel). The insert in the top panel is the similarity matrix showing the pairwise chi2 between the SAXS profiles of each PDZ1-2 construct (for q ranging from 0.119 to 4.007 nm-1).
(C) Radius of gyration (Rg) and maximum distance (Dmax) values in Ångströms extracted from the scattering curve of each PDZ1-2 construct. It shows two families of constructs, one more compact (PDZ1-2WT, PDZ1-2W375G, and PDZ1-2IP225GG, highlighted in blue) and the other one more extended (PDZ1-2W237G and PDZ1-2LP3635GG, highlighted in red).
(D) Distance distribution function extracted from the SAXS data of the various constructs.
(E) Fluorescence emission spectra measured for PDZ1-2 wild-type and mutants, monitoring the local environment of the tryptophans located in the hairpin extensions of each PDZ domain.
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5. Figure 3
Correlation between the Structural Ensemble and the Experimental PRE and RDC Used as Restraints in the Docking Calculation
(A) Experimental ratio of cross-peak intensities in the spectra corresponding to oxidized and reduced forms (gray bars) and back-calculated ratios derived from the structural ensemble of the 25 models of lowest energy (averages and SDs as blue dots and lines) for spin labels attached to four different positions indicated as spheres (E162C in orange, T237C in yellow, S290C in dark green, and D371C in light green).
(B) Correlation plots between experimental 1N-15N RDC and back-calculated RDC derived from the structural ensemble of the 25 best models (top panel, phages; bottom panel, ether/alcohol).
(C) Quality score between the experimental and back-calculated cross-peak intensity ratio from the structural ensemble for all intra- or inter-domain PRE restraints and for each individual set from different probe positions.
(D) Root-mean-square difference between experimental and back-calculated RDC from the structural ensemble for the two alignment media.
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6. Figure 4 Structural Model of the Whirlin PDZ1-2 Supramodule
(A–C) Cartoon representation of the whirlin PDZ1-2 structural model in different orientations. The closest structure to the ensemble average of the 25 models is represented. PDZ1 is colored in orange with its associated hairpin in yellow, and PDZ2 is represented in green, with its hairpin in light green. The spherical residues are the four residues substituted with cysteine for paramagnetic labeling (color coding as in Figure 3A).
(D) Superimposition of the 25 models of lowest energy on the rigid secondary structures of both domains.
(E and F) Volume representation of the whirlin PDZ1-2 structural model showing the accessibility of the binding site (light blue) and the “GLGF” loop (dark blue) of the two PDZ domains.
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7. Figure 5
Comparison of PDZ1-2 Mutation Effects with the PDZ1-2 Supramodule Model
(A) Triplicate average of differential fractional uptake of deuterium between the hairpin mutants and wild-type PDZ1-2 monitored by mass spectrometry after increasing the duration of incubation (10, 60, 600, 1,800, and 3,600 s). Peptides containing the mutations are indicated by downward triangles. Missing peptides induced by changes of enzymatic proteolysis in mutants are indicated by black segments. Peptides with significantly modified uptake are highlighted in red on the graph and the x axis.
(B) NMR chemical shift perturbations (gray bars) and peak intensity ratio (violet dots) between 1H-15N HSQC spectra of hairpin mutants and wild-type PDZ1-2. Spheres using the mutant color coding of Figure 2A show mutation positions. The analysis of perturbations induced in compact mutants (blue) and extended mutants (red) delineates the PDZ interface (red transparent boxes).
(C) The whirlin PDZ1-2 structural model with the hairpin substitutions PDZ1-2IP225GG, PDZ1-2W237G, PDZ1-2LP363GG, PDZ1-2W375G shown as spheres using the color coding of Figure 2A.
(D) Mapping (dark red spheres) of the increase in deuterium uptake observed by H/D exchange in (B) of the “open” tandem PDZ1-2W237G and PDZ1-2LP363GG onto the PDZ1-2 structural model. The PDZ1-2W237G and PDZ1-2LP363GG mutations are shown as light (W237G) and bright red (L363G-P364G) spheres, respectively.
(E) Mapping onto the whirlin PDZ1-2 structural model of the residues affected by inter-domain CSP and intensity changes (dark red spheres) observed with the PDZ1-2W237G substitution (in light red spheres).
(F) Mapping onto the whirlin PDZ1-2 structural model of the residues affected by inter-domain CSP and intensity changes (dark red spheres) observed with the PDZ1-2H340A substitution (in black).
(G) NMR chemical shift perturbations (gray bars) induced by the H340A mutation in PDZ2 (black sphere). Inter-domain perturbations on PDZ1 and hairpin1 (highlighted in red) are due to the opening of the mutant compared with the wild-type and are in perfect agreement with the structural model.
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8. Figure 6 Selection of Conformations Describing PDZ1-2 Equilibrium
(A) Fit improvement in chi2 according to the ensemble size in the genetic algorithm selection for the SAXS data when the selection is performed individually (dotted blue line) or combined with the RDC dataset (continuous blue line).
(B) Fit improvement in the Q factor according to the ensemble size in the genetic algorithm selection for RDC ether/alcohol mixture and phage medium, when the selection is performed individually (dotted orange and green lines, respectively) or combined with the RDC and SAXS dataset (plain orange and green lines, respectively). The error bars are computed over ten individuals run.
(C) Left panel: back-calculated SAXS profile derived from the best ensemble (100 conformations preset, with 18 unique conformations selected, in blue) and the experimental profile (black dots). Middle panel and right panel: correlation between the back-calculated RDC from this ensemble and the experimental RDC from an ether/alcohol mixture and phage medium (middle and right, respectively). The chi2 and Q factors are indicated.
(D) The conformations are clustered in two categories according to their RMSD with the NMR-derived structural model; conformations with RMSD lower than 10 Å are considered “closed” (green bars) while the others are considered “open” (violet bars). The proportion of each cluster is represented on a stacked bar chart for each ensemble size selected on the wild-type SAXS and RDC dataset or on the PDZ1-2LP363GG SAXS dataset.
(E) Representative ensemble of PDZ1-2 superimposed on PDZ1 (orange) showing the PDZ2 in compact conformation (green) or in extended conformation (violet) with the proportion of each conformer. Only the centroids of the clusters obtained on the selected ensemble is shown.
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9. Figure 7 Interaction between Whirlin and the Peptide Sans
(A) Table of affinity between Sans peptide and different constructs of whirlin and a plot showing the decrease in dissociation constants according to the whirlin constructs. The average and SDs are calculated on ten significantly shifted peaks.
(B) 1H-15N HSQC spectra of the titration of PDZ1-Hp1 with Sans peptide. The PDZ1-Hp1 without peptide is shown in blue and the last point of the titration in red. The eight intermediate points are shown in shades of gray. Examples of residues affected by peptide binding in hairpin1 are shown in boxes.
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