Volume 20, Issue 7, Pages (July 2012)

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Volume 20, Issue 7, Pages 1212-1222 (July 2012) Structural Insights into the Role of Domain Flexibility in Human DNA Ligase IV  Takashi Ochi, Qian Wu, Dimitri Y. Chirgadze, J. Günter Grossmann, Victor M. Bolanos-Garcia, Tom L. Blundell  Structure  Volume 20, Issue 7, Pages 1212-1222 (July 2012) DOI: 10.1016/j.str.2012.04.012 Copyright © 2012 Elsevier Ltd Terms and Conditions

Structure 2012 20, 1212-1222DOI: (10.1016/j.str.2012.04.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 Gel Filtration Chromatography Studies of Complex Formation of LigIV, XRCC4ΔCTD;CtoA, and XLFΔCTD (A) Profiles of the UV absorbance at 280 nm during gel filtration chromatography. Colors of profiles and their corresponding constructs are shown at the bottom of the figure. Gray arrows indicate peak positions of protein standards, void, ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), and RNase A (13.7 kDa). (B) SDS-PAGE of LX4ΔCTD;CtoA and XLFΔCTD fractions eluted from a Superdex 200 10/30 column. The molecular weight markers are in column “M” column and their molecular weights (kDa) are shown on the left of the gel. The fraction ranges used for the SDS-PAGE are indicated using a blue arrow in (A). Each fraction contained 250 μl of the eluted sample. (C) SDS-PAGE of LΔcatX4ΔCTD;CtoA and XLFΔCTD eluted from the gel filtration column. The fractions used for SDS-PAGE are indicated alphabetically (green a-n) both in (A) and in the gel. (D) Schematic representation of the constructs used in the gel filtration experiment. The domain names and boundaries are shown in LX4ΔCTD;CtoA and XLFΔCTD. In XRCC4ΔCTD;CtoA and XLFΔCTD, HD, CC, and FB represent head, coiled-coil, and fold-back domains, respectively. See also Figure S1. Structure 2012 20, 1212-1222DOI: (10.1016/j.str.2012.04.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 SAXS Studies of LX4 (A) Experimental scattering curves of LX4 constructs. The scattering intensities (log I versus s-value) with error bars (gray) of LX4 (blue), LX4ΔCTD;CtoA (green) and LΔcatX4ΔCTD;CtoA (blue) are displaced by factor of 100 for clarity. The scattering curves of the latter two constructs were modified after Ochi et al., 2010. (B) Distance distributions of LX4 constructs. The same color scheme as in (A) is used in this figure. The error bars are represented with gray. (C) Shape reconstruction of LX4ΔCTD;CtoA. The molecular envelope of LX4ΔCTD;CtoA is shown in two perpendicular orientations, which derived from an averaging process of several, individually restored 3D shapes. The structure of a LX4 construct (PDB code: 3II6; Wu et al., 2009). The structure was fitted into the envelope manually and refined using Chimera (Pettersen et al., 2004). The two structural superimpositions providing the highest correlation correlations coefficients are illustrated to highlight additional molecular density not present in the crystal structure. See also Figure S2. Structure 2012 20, 1212-1222DOI: (10.1016/j.str.2012.04.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 Structure of NTase-3 (A) Overall architecture of NTase-3. Conserved motifs I, III and IIIa are shown in purple, pink and yellow. Dotted lines represent missing loops (gray and pink) connecting DNA-binding regions D1 and D2 (pink). (B) A schematic presentation of secondary structure elements of NTase-3. See also Figure S3. Structure 2012 20, 1212-1222DOI: (10.1016/j.str.2012.04.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 Rigid-Body Modeling and Protein-Protein Binding Assays of Human DNA Ligase IV/XRCC4 Complex (A) Rigid-body modeling of LX4ΔCTD;CtoA using BUNCH. Ten individual rigid-body models were superposed on the structure of the LΔcatX4ΔCTD;CtoA region. The models with the three highest χ2 values are shown in a cartoon representation and the others are shown as their Cα traces. (B) Left: EMSAs of individual catalytic domains and LΔcatX4. The proteins used are indicated with “+.” Right: GST pull-down assays of OBD and LΔcatX4. The upper and lower figures show the results of the assays using GST-OBD-620 and GST-OBD-653, respectively. The first lane protein markers (M) are followed by unbound proteins (U) and bound proteins (B) to GST affinity resin. Structure 2012 20, 1212-1222DOI: (10.1016/j.str.2012.04.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 5 Comparison of the DNA-Binding Loop D1 of NTase-3 The structure of NTase-3 of LigIV (cyan) is shown together with that of LigI (I, pink; PDB code: 1X9N) and LigIII (III, blue; PDB code: 3L2P), and DNA (PDB code: 1X9N). Backbone phosphates of DNA are labeled as 12 and 13. The pink-dotted lines represent hydrogen bonds between LigI and the DNA. The original residue numbers of the phosphates shown in the PDB file are used here. See also Figure S4. Structure 2012 20, 1212-1222DOI: (10.1016/j.str.2012.04.012) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 6 Interactions of Residues that Cause LIG4 Syndrome that Are Close to the Catalytic Residues of Human LigIV (A) Hydrophobic core of NTase-3 of LigIV around Y288 (magenta). Since the electron density for the side chains of K283 and I315 was not observed, the amino acids were represented as alanines. (B) Interactions in the model between R278 (magenta) and surrounding residues. (C) Interactions between Q280/H282 and surrounding residues. Salt bridges and hydrogen bonds are represented using black dotted lines. See also Figure S5. Structure 2012 20, 1212-1222DOI: (10.1016/j.str.2012.04.012) Copyright © 2012 Elsevier Ltd Terms and Conditions