Volume 23, Issue 4, Pages 724-733 (April 2015) Structurally Distinct Ubiquitin- and Sumo-Modified PCNA: Implications for Their Distinct Roles in the DNA Damage Response  Susan E. Tsutakawa, Chunli Yan, Xiaojun Xu, Christopher P. Weinacht, Bret D. Freudenthal, Kun Yang, Zhihao Zhuang, M. Todd Washington, John A. Tainer, Ivaylo Ivanov  Structure  Volume 23, Issue 4, Pages 724-733 (April 2015) DOI: 10.1016/j.str.2015.02.008 Copyright © 2015 Elsevier Ltd Terms and Conditions

Structure 2015 23, 724-733DOI: (10.1016/j.str.2015.02.008) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 Distinct Architectures of PCNA-Ub and PCNA-SUMO Complexes from SAXS Analysis (A) SAXS profiles of PCNAK164-SUMO (blue), PCNAK107-Ub (green), and PCNAK164-Ub (red). (B) P(r) functions for PCNAK164-SUMO (blue), PCNAK107-Ub (green), and PCNAK164-Ub (red). (C) Guinier analyses of SAXS data for PCNAK107-Ub (green) and PCNAK164-SUMO (blue) showing relative linearity of the samples in the Guinier region, indicating lack of aggregation. (D) A dimensionless Kratky plot indicates PCNAK164-SUMO being significantly less compact than PCNAK164-Ub, which is slightly less compact than PCNAK107-Ub (green). All P(r) distributions were normalized by dividing the values by the peak height. Structure 2015 23, 724-733DOI: (10.1016/j.str.2015.02.008) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 2 Rosetta Score versus RMSD Plots for Ubiquitinated and SUMOylated PCNA (A) Decoys from PCNAK107-Ub docking are shown in gray. Lowest-scoring structurally distinct models (selected for building triplets) are shown in red. One model (blue dot) departed from its binding position during molecular dynamics (MD) and was not considered for minimal ensemble search (MES) analysis. (B) Decoys from PCNAK164-SUMO docking are shown in gray. Lowest-scoring models (selected for building triplets) are shown in red. Four models (blue dots) departed during MD and were not considered for MES analysis. One partially flexible position (purple) was subsequently included in MES analysis. Structure 2015 23, 724-733DOI: (10.1016/j.str.2015.02.008) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 3 Ub Primarily Adopts Docked Positions in PCNAK107-Ub while SUMO Occupies Extended Positions in PCNAK164-SUMO An MES produces the best fit to the experimental SAXS data for PCNAK107-Ub and PCNAK164-SUMO. (A) χ values for the triplet PCNAK107-Ub structures plotted against RMSD. Conformations selected by MES are highlighted in blue and magenta, respectively. (B) χ values for the triplet PCNAK164-SUMO structures plotted against RMSD. Conformations selected by MES are highlighted in blue, magenta, and red, respectively. (C) Overlaid experimental SAXS profile for PCNAK107-Ub (green), computed profile for 3L10 crystal structure (red dashed line), and computed profile from MES model (black). (D) Overlaid experimental SAXS profile for PCNAK164-SUMO (blue), computed profile for 3V60 crystal structure (red dashed line), and computed profile from MES model (black). (E) P(r) functions for PCNAK107-Ub (green), 3L10 crystal structure (red dashed line), and MES model (black). (F) P(r) functions for PCNAK164-SUMO (blue), 3V60 crystal structure (red dashed line), and MES model (black). (G) The two most populated atomic structures from MES analysis of PCNAK107-Ub in surface representation. (H) The three most populated atomic structures from MES analysis of PCNAK164-SUMO in surface representation. The P loop is shown in purple; the K107 and K164 attachment points are depicted in red. PCNA, Ub, and SUMO are shown in gray, green, and blue, respectively. The MES occupancies for the three conformations are labeled in blue, magenta and red, respectively. All P(r) distributions were normalized by dividing the values by the peak height. Structure 2015 23, 724-733DOI: (10.1016/j.str.2015.02.008) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 4 Structural Differences in PCNAK107-Ub and PCNAK164-SUMO Complexes from the Anticorrelated Electrostatic Potential of Ub and SUMO (A) Ub docked onto PCNA in the most populated MES positions (P-loop, subunit interface, and central cavity positions) and SUMO position from the 3V60 crystal structure. Ub, SUMO, and PCNA are colored green, blue, and gray, respectively. The interdomain connector loops (IDCL) and P loops on PCNA are shown in orange and purple, respectively. The attachment positions, K107 and K164 residues, are displayed as red spheres. (B) Electrostatic potential surfaces corresponding to the bound positions of Ub and SUMO on PCNA (P loop, subunit interface, and central cavity, from left to right for PCNAK107-Ub) and 3V60 structure for PCNAK164-SUMO. NaCl (100 mM) was introduced to screen the electrostatics mimicking physiological conditions. The potential varies from −5KBT/e to +5KBT/e and is depicted from red to blue, respectively. Structure 2015 23, 724-733DOI: (10.1016/j.str.2015.02.008) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 5 Distinct Types of Interfaces Are Exposed Depending on the Different Modes of Association of Ubiquitin and SUMO to PCNA The PCNA trimer is shown in gray, the Ub modifier in green, and SUMO in blue; the attachment positions are indicated by red dots; curved arrows indicate flexible attachment. Approximate occupancy (%) of the identified distinct positions of the modifiers on PCNA is given below each model. Structure 2015 23, 724-733DOI: (10.1016/j.str.2015.02.008) Copyright © 2015 Elsevier Ltd Terms and Conditions