Volume 24, Issue 12, Pages 2053-2066 (December 2016) Distinct Roles for Conformational Dynamics in Protein-Ligand Interactions  Xu Liu, David C. Speckhard, Tyson R. Shepherd, Young Joo Sun, Sarah R. Hengel, Liping Yu, C. Andrew Fowler, Lokesh Gakhar, Ernesto J. Fuentes  Structure  Volume 24, Issue 12, Pages 2053-2066 (December 2016) DOI: 10.1016/j.str.2016.08.019 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 2053-2066DOI: (10.1016/j.str.2016.08.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Design of the QM PDZ Domain (A) Space-filling model of the Tiam1 PDZ in complex with SDC1 peptide ligand (PDB: 4GVD). Residues that form the S0 and S-2 specificity pockets are labeled and colored in red. (B) Primary sequence alignment of the human Tiam1, QM, and mouse Tiam2 PDZ domains. Secondary structure is indicated by rectangles (α helix) and arrows (β strand). The six residues labeled in (A) are highlighted in the sequence alignment. The four mutations in the QM are colored yellow. An asterisk represents conserved residues; a colon represents conservation between residues with strongly similar properties; a period represents conservation between residues with weakly similar properties. Structure 2016 24, 2053-2066DOI: (10.1016/j.str.2016.08.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Chemical Shift Changes in the QM PDZ Domain Overlaid 1H-15N HSQC spectra of QM (blue) and Tiam1 PDZ (red) domains. Residues with the largest changes in chemical shift are labeled and indicated by arrows. See also Figures S1 and S3. Structure 2016 24, 2053-2066DOI: (10.1016/j.str.2016.08.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 The QM PDZ Domain Has Switched Ligand Binding Specificity (A) Representative binding curves for the interaction between the QM PDZ domain and dansylated peptides derived from the syndecan family (SDC1-4), Caspr4, neurexin1 (NRXN1), and CADM1. (B) Summary of QM PDZ/peptide binding data. The sequence of each peptide and the determined dissociation constant (Kd) is indicated. Fold change represents Kd(WT)/Kd(QM). The Kd values reflect the mean and SD from at least three technical replicates. See also Figure S3 and Table S1. Structure 2016 24, 2053-2066DOI: (10.1016/j.str.2016.08.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 Thermodynamic Analysis of the QM PDZ/Caspr4 Domain Interaction by ITC (A) Thermogram and integrated titration curve for the Caspr4 ligand bound to the QM PDZ domain. (B) Thermodynamic parameters for the Tiam1 PDZ/Caspr4 and QM PDZ/Caspr4 interactions at 298 K. Each parameter represents the mean and SD from three technical replicates. See also Table S5. Structure 2016 24, 2053-2066DOI: (10.1016/j.str.2016.08.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Structures of the QM PDZ Domain Free and Bound to Caspr4 and NRXN1 Peptides (A) Ribbon representation of the QM PDZ (pink) and Tiam1 PDZ (PDB: 3KZD) (gray) domains. Side chains of the four residues mutated in this study are labeled in red and shown as sticks. The dashed line represents residues without interpretable electron density in both structures. (B) Structural model of the QM PDZ/Caspr4 complex showing backbone and side-chain interactions. PDZ domain residues involved in peptide binding are colored yellow and labeled, while the Caspr4 peptide is colored cyan. The zoomed-in view on the right shows several unique interactions denoted by dotted lines. The conformation of the four mutated residues in the apo QM (pink) and Caspr4-bound (yellow) structures are shown for comparison. (C) Structural model of the QM PDZ/NRXN1 complex showing backbone and side-chain interactions. PDZ domain residues involved in peptide binding are colored yellow and labeled, while the NRXN1 peptide is colored green. The zoomed-in view on the right shows several interactions and the conformation of the four mutated residues in the apo QM (pink) and NRXN1-bound (yellow) structures. See also Figure S2 and Table S2. Structure 2016 24, 2053-2066DOI: (10.1016/j.str.2016.08.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 Fast, Picosecond to Nanosecond, Timescale Backbone Dynamics of the QM PDZ Domain (A and B) The order parameter (S2), timescale of motion (τe, ●), and chemical exchange (Rex, Δ) of the free QM PDZ domain is plotted against backbone amide residue. The secondary structure is shown on the top of the graph. Regions with enhanced dynamics are shaded in gray. (C and D) The changes of order parameter (ΔS2, ○) and chemical exchange (ΔRex, Δ) in the Tiam1 PDZ (WT) domain caused by the four mutations (C) and those in the QM PDZ domain caused by Casrpr4 binding (D). Error bars for each parameter represent the propagated uncertainty determined from Monte Carlo simulations. Symbols for residues that experience significant changes in a particular parameter (>2-fold the propagated error) are colored black in (C) and (D). An asterisk indicates that the data for either the free or bound state was analyzed using a dynamic model that did not include a Rex term. See also Figures S4 and S5. Structure 2016 24, 2053-2066DOI: (10.1016/j.str.2016.08.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 Fast, Picosecond to Nanosecond, Timescale Methyl-bearing Side-Chain Dynamics in the QM PDZ Domain (A–C) The change in S2axis and τe in the QM PDZ domain compared with the Tiam1 PDZ domain (WT). (D–F) The change in S2axis and τe in the QM PDZ domain upon binding Caspr4. Black colored bars indicate residues that experience significant (>2-fold the propagated error) changes in this parameter. The error bars represent propagated uncertainty as derived from Monte Carlo simulations. Methyl groups exhibiting changes in dynamics are mapped onto structural models (C) and (F) of the free and Caspr4-bound QM PDZ domains, respectively. The methyl groups (spheres) are colored in a continuous gradient from red to blue, with their intensity scaling to the magnitude of ΔS2axis. The Caspr4 peptide is shown in cyan. Methyl groups that had a significant Δτe but no ΔS2axis are shown as spheres and colored yellow. Residues Y858, F860, M911, E912, F915, and V920 are shown as sticks and colored yellow. See also Tables S3–S5. Structure 2016 24, 2053-2066DOI: (10.1016/j.str.2016.08.019) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 8 Slow, Microsecond to Millisecond, Timescale Motions in the QM PDZ Domain (A and B) Representative CPMG relaxation dispersion curves are shown for the Tiam1 and QM PDZ domains, respectively. Individual curves for each residue with Rex are shown in Figure S6, while their fitted parameters are indicated in Table 2. Data collected at 800 MHz (closed circle) and 500 MHz (open circle) are shown. Error bars were determined by the analysis of peak intensities from duplicate experiments. (C) Residues with Rex in the QM PDZ domain are labeled and colored in red. The six residues shown in Figure 1 have their side chains displayed. Those colored yellow did not have Rex. See also Figures S4 and S5. Structure 2016 24, 2053-2066DOI: (10.1016/j.str.2016.08.019) Copyright © 2016 Elsevier Ltd Terms and Conditions