Structurally distinct ubiquitin- and sumo-modified PCNA: implications for their distinct roles in the DNA damage response

Abstract

Proliferating cell nuclear antigen (PCNA) is a pivotal replication protein, which also controls cellular responses to DNA damage. Posttranslational modification of PCNA by SUMO and ubiquitin modulate these responses. How the modifiers alter PCNA-dependent DNA repair and damage tolerance pathways is largely unknown. We used hybrid methods to identify atomic models of PCNAK107-Ub and PCNAK164-SUMO consistent with small-angle X-ray scattering data of these complexes in solution. We show that SUMO and ubiquitin have distinct modes of association to PCNA. Ubiquitin adopts discrete docked binding positions. By contrast, SUMO associates by simple tethering and adopts extended flexible conformations. These structural differences are the result of the opposite electrostatic potentials of SUMO and Ub. The unexpected contrast in conformational behavior of Ub-PCNA and SUMO-PCNA has implications for interactions with partner proteins, interacting surfaces accessibility, and access points for pathway regulation.

Figure 1. Distinct architectures of PCNA-Ub and PCNA-SUMO complexes from SAXS analysis

(A) SAXS profiles of PCNA_K164-SUMO (blue), PCNA_K107-Ub (green) and PCNA_K164-Ub (red); (B) P(r) functions for PCNA_K164-SUMO (blue), PCNA_K107-Ub (green) and PCNA_K164-Ub (red); (C) Guinier analyses of SAXS data for PCNA_K107-Ub (green) and PCNA_K164-SUMO (blue) showing relative linearity of the samples in Guinier region, indicating lack of aggregation. (D) A dimensionless Kratky plot indicates PCNA_K164-SUMO being significantly less compact than PCNA_K164-Ub, which is slightly less compact than PCNA_K107-Ub (green). All P(r) distributions were normalized dividing the values by the peak height.

Figure 2. Rosetta score versus RMSD plots for ubiquitinated and SUMOylated PCNA

(A) Decoys from PCNA_K107-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 MD and was not considered for MES analysis. (B) Decoys from PCNA_K164-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.

Figure 3. Ub primarily adopts docked positions in PCNA_K107-Ub while SUMO occupies extended positions in PCNA_K164-SUMO

A minimal ensemble search (MES) produces the best fit to the experimental SAXS data for PCNA_K107-Ub and PCNA_K164-SUMO. (A) χ values for the triplet PCNA_K107-Ub structures plotted against RMSD. Conformations selected by MES are highlighted in blue and magenta, respectively. (B) χ values for the triplet PCNA_K164-SUMO structures plotted against RMSD. Conformations selected by MES are highlighted in blue, magenta and red, respectively. (C) Overlaid experimental SAXS profile for PCNA_K107-Ub (green), computed profile for 3L10 crystal structure (red dotted line) and computed profile from MES model (black). (D) Overlaid experimental SAXS profile for PCNA_K164-SUMO (blue), computed profile for 3V60 crystal structure (red dotted line) and computed profile from MES model (black). (E) P(r) functions for PCNA_K107-Ub (green), 3L10 crystal structure (red dotted line) and MES model (black). (F) P(r) functions, for PCNA_K164-SUMO (blue), 3V60 crystal structure (red dotted line) and MES model (black). (G) The two most populated atomic structures from MES analysis of PCNA_K107-Ub in surface representation. (H) The three most populated atomic structures from MES analysis of PCNA_K164-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 diving the values by the peak height.

Figure 4. Structural differences in PCNA_K107-Ub and PCNA_K164-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 IDCL and P-loops on PCNA are shown in orange and purple, respectively. The attachment positions, K107 and K164 residues, are displayed as red balls. (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 PCNA_K107-Ub) and 3V60 structure for PCNA_K164-SUMO). 100 mM NaCl concentration was introduced to screen the electrostatics mimicking physiological conditions. The potential varies from −5K_BT/e to +5K_BT/e and is depicted from red to blue, respectively.

Figure 5. Distinct types of interfaces are exposed dependent on the different mode of association of ubiquitin and SUMO to PCNA

The PCNA trimer is shown in gray; the Ub modifier in green; SUMO in blue; the attachment positions are indicated by a red dots; curved arrows indicate flexible attachment. Approximate occupancy (%) of the identified distinct positions of the modifiers on PCNA given below each model.