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Authors
Susan E. Tsutakawa, Chunli Yan, ...,
John A. Tainer, Ivaylo Ivanov
Correspondence
iivanov@gsu.edu (I.I.),
jatainer@lbl.gov (J.A.T.)
In Brief
Tsutakawa et al. combine computational
modeling and small-angle X-ray
scattering to show that SUMOylated and
ubiquitinated PCNA have strikingly
different conformations in solution due to
opposite electrostatics of the modifiers.
The distinct conformations underlie
distinct roles for PCNA-Ub and PCNASUMO in DNA damage responses.
Highlights
d
Please cite this article in press as: Tsutakawa et al., Structurally Distinct Ubiquitin- and Sumo-Modified PCNA: Implications for Their Distinct Roles in
the DNA Damage Response, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.02.008
Structure
Article
Structurally Distinct Ubiquitin- and Sumo-Modified
PCNA: Implications for Their Distinct
Roles in the DNA Damage Response
Susan E. Tsutakawa,1,7 Chunli Yan,2,7 Xiaojun Xu,2 Christopher P. Weinacht,3 Bret D. Freudenthal,4,8 Kun Yang,3
Zhihao Zhuang,3 M. Todd Washington,4 John A. Tainer,1,5,6,* and Ivaylo Ivanov2,*
1Life
SUMMARY
INTRODUCTION
Dynamic assembly, coordinated access to DNA, and conformational switching are critical aspects of DNA replication and
repair, essential processes upon which life depends. In both processes, the sliding clamp proliferating cell nuclear antigen
(PCNA) (Ivanov et al., 2006; Kelman, 1997; Krishna et al., 1994;
Moldovan et al., 2007) acts as a master coordinator of multiple
pathways controlling replication and DNA damage responses
(DDR). The toroidal shape of PCNA allows it to topologically
encircle DNA while also binding to core replisomal constituents
and numerous repair and cell-cycle control proteins (Jonsson
Structure 23, 110, April 7, 2015 2015 Elsevier Ltd All rights reserved 1
Please cite this article in press as: Tsutakawa et al., Structurally Distinct Ubiquitin- and Sumo-Modified PCNA: Implications for Their Distinct Roles in
the DNA Damage Response, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.02.008
receptor motifs: a SUMO interaction motif (SIM) and a noncanonical PIP box (Armstrong et al., 2012; Freudenthal et al.,
2011). Both motifs are required for specific recognition of
SUMO-PCNA. One possible mechanism for Srs2 to suppress
homologous recombination is through disruption of Rad51 filaments (Krejci et al., 2003; Veaute et al., 2003), thereby assisting
the ubiquitin-dependent TLS pathway. A novel alternative mechanism has been proposed wherein Srs2 binding to SUMO-PCNA
dissociates the replicative and TLS polymerases from the repair
synthesis machinery and, thus, prevents the synthesis dependent extension of recombination intermediates (Burkovics
et al., 2013). This latter mechanism requires only Srs2 recruitment through the SIM motif but neither its translocase
activity nor interaction with Rad51. PCNA ubiquitination and
SUMOylation offer striking examples of crosstalk between pathways controlled by distinct PTMs (Stelter and Ulrich, 2003; Ulrich, 2005). While attachment of a single ubiquitin is the dominant
DDR response in mammalian cells, yeast PCNA can also undergo polyubiquitination through the non-proteasomal K63 linkage (Hoege et al., 2002). Polyubiquitination of PCNA at K164
channels DDR to yet another pathway, error-free damage
bypass by template switching. In addition, in yeast PCNA monoubiquitination at position K107 helps cells to overcome defects in
DNA ligation through an S-phase checkpoint activation (DasBradoo et al., 2010; Nguyen et al., 2013). Damage-related ubiquitination at other PCNA positions has been recently identified:
PCNA was ubiquitinated at K248 after UV irradiation (Povlsen
et al., 2012) and ubiquitinated at K168 after colchicine treatment
(Xu et al., 2010). How could PTMs introduced at different positions on PCNA result in such vastly divergent functional outcomes? Furthermore, how do posttranslational modifications
of PCNA facilitate recruitment of subsequent effector proteins
in these pathways?
To answer these questions, we modeled PCNA covalently
modified by ubiquitin at residue K107 and SUMO at K164
using a multiscale computational protocol. Models consistent
with solution X-ray scattering data were identified (Hura et al.,
2009; Putnam et al., 2007; Rambo and Tainer, 2010, 2011),
which allowed us to assess the structural differences of PTMPCNA complexes (PCNAK107-Ub and PCNAK164-SUMO) and
compare these with PCNAK164-Ub from a previous study. Here
we show that SUMO and Ub have distinct modes of interaction
with PCNA and that the position of ubiquitin attachment, 107
versus 164, alters conformation. Ubiquitin bound to PCNA can
dynamically adopt multiple discrete docked conformations. By
contrast, SUMO is flexibly tethered, with no substantial docking
interactions with PCNA. Our hybrid structural analysis reveals
the biologically relevant conformations of modified PCNA in
solution that effector proteins would first encounter and interact
with.
RESULTS
Distinct Architectures of Modified PCNA Complexes
Uncovered by SAXS Data
We conducted small-angle X-ray scattering (SAXS) experiments
to probe the overall architecture and flexibility of Saccharomyces cerevisiae PCNAK107-Ub and PCNAK164-SUMO complexes in solution. Covalent attachment in the PCNA-Ub com-
2 Structure 23, 110, April 7, 2015 2015 Elsevier Ltd All rights reserved
Please cite this article in press as: Tsutakawa et al., Structurally Distinct Ubiquitin- and Sumo-Modified PCNA: Implications for Their Distinct Roles in
the DNA Damage Response, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.02.008
Structure 23, 110, April 7, 2015 2015 Elsevier Ltd All rights reserved 3
Please cite this article in press as: Tsutakawa et al., Structurally Distinct Ubiquitin- and Sumo-Modified PCNA: Implications for Their Distinct Roles in
the DNA Damage Response, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.02.008
c and cfree (Rambo and Tainer, 2013) values were used to measure goodness of fit of the computed profiles to the experimental
scattering data. Computed c values for PCNAK107-Ub and
PCNAK164-SUMO are plotted in Figures 3A and 3B as a function
of Ca RMSD for each conformation. The single best fit to the
experimental data was c = 0.78 (cfree = 0.82) for PCNAK107-Ub
and c = 2.55 (cfree = 2.97) for PCNAK164-SUMO, respectively.
However, in a flexible system multiple conformations will
contribute to the SAXS profile. Therefore, a minimal ensemble
search (MES) (Pelikan et al., 2009) was used to identify a small
subset of conformations that optimally represent the scattering
data. In the case of PCNAK107-Ub, an excellent agreement to
the experimental curve was obtained (c = 0.74; cfree = 0.78) (Figure 3C; Figure S3A) by a set of two distinct conformers with
ensemble contributions of 59% and 41% (Figure 3G). In these
conformers the Ub occupied primarily docked positions, closely
associating with the PCNA surface. In the case of PCNAK164SUMO, however, the ensemble was dominated by two flexible,
extended conformations (45% and 31% occupancy) and a third
flexible but more compact conformation (24% occupancy) (Figure 3H). MES notably improved the goodness of fit to the PCNA-
SUMO SAXS data (c = 2.16, cfree = 2.41, Figure 3D; Figure S3B).
Conversely, large discrepancies between the crystal structure
3V60 and the solution SAXS profile were observed (c = 6.70,
cfree = 7.65, Figure 3B), indicating that the conformation found
in the crystal is not prevalent in the solution phase ensemble.
In all models selected by MES, we calculated the occupancy
of docked versus extended flexible positions for PCNAK107-Ub
and PCNAK164-SUMO. In the PCNAK107-Ub complex, the ubiquitins were all positioned on the back side of PCNA. Approximately
half of the Ub population was docked along the P loop, similar to
the position observed in the crystal structure. One-third was at
the subunit interface. The remainder (20%) was located near
the central cavity. All three positions allow dsDNA to pass
through PCNA and, intriguingly, would place Ub in proximity to
the dsDNA backbone passing through PCNA (Figure S4). The
attachment position at K107 is on the back face of the clamp
close to the subunit interface. Thus, switching the point of
attachment from K164 to K107 leads to an increased probability
to locate the Ub moiety on the back face of PCNA. Unlike the
PCNAK164-Ub study, the MES did not identify any flexible conformations. The goodness of the fit of these back positions to the
experimental SAXS data was consistent with this observation.
In the case of PCNA-SUMO, extended positions were identified in the MES calculation. As shown in Figure 3D, MES analysis
identifies SUMO with 84% occupancy in two extended flexible
positions. Interestingly, MES did not pick any positions docked
to the PCNA surface despite inclusion of the 3V60 crystallographic position in the MES optimization. This outcome indicates
that SUMO is largely flexible in solution. It is notable that the
docked positions observed by crystallography (Armstrong
et al., 2012; Freudenthal et al., 2011) (3PGE and 3V60) have
significant crystallographic contacts with few intramolecular
contacts.
Detailed Interactions in the MES Identified Models
To identify specific structural features that might drive the closer
association of Ub to PCNA, we inspected the two most populated MES triplet models. The primary position near the P loop
was originally observed in the crystal structure and is based on
a hydrophobic contact of the major protein interacting face
on Ub, Ile44 Ub and Val70Ub with Met188PCNA and Val186PCNA.
The Ub position above the subunit interface was stabilized
through several salt bridges: Arg42Ub-Glu7PCNA, Lys48UbGlu59PCNA, Glu51Ub-Lys5PCNA, and Glu58Ub-Arg61PCNA. The
position near the central cavity had two salt bridges, Asp39UbK107PCNA and Asp58Ub-Lys242PCNA. A stacking of aliphatic
chains between Arg72Ub-Asp109PCNA is also observed at the
interface between PCNA and Ub. This Ub position could also
be explained by a general electrostatic attraction between the
acidic face of ubiquitin moving toward the basic central cavity.
It is notable in the last two positions that Ile44Ub and Val70Ub
are exposed, perhaps promoting an interface for protein-protein
interaction with Ub.
In contrast to the ubiquitin-modified PCNA, our MES results
with SUMOylated PCNA suggest that covalently attached
SUMO actually makes few contacts to PCNA in solution and
adopts extended flexible positions. The crystal structure of
PCNAK164-SUMO revealed that the interface with the modifier
comprises loop regions of PCNA and SUMO, which involve
4 Structure 23, 110, April 7, 2015 2015 Elsevier Ltd All rights reserved
Please cite this article in press as: Tsutakawa et al., Structurally Distinct Ubiquitin- and Sumo-Modified PCNA: Implications for Their Distinct Roles in
the DNA Damage Response, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.02.008
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) c values for the triplet PCNAK107-Ub structures plotted against RMSD. Conformations selected by MES are highlighted in blue and magenta, respectively.
(B) c 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 23, 110, April 7, 2015 2015 Elsevier Ltd All rights reserved 5
Please cite this article in press as: Tsutakawa et al., Structurally Distinct Ubiquitin- and Sumo-Modified PCNA: Implications for Their Distinct Roles in
the DNA Damage Response, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.02.008
6 Structure 23, 110, April 7, 2015 2015 Elsevier Ltd All rights reserved
Please cite this article in press as: Tsutakawa et al., Structurally Distinct Ubiquitin- and Sumo-Modified PCNA: Implications for Their Distinct Roles in
the DNA Damage Response, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.02.008
Structure 23, 110, April 7, 2015 2015 Elsevier Ltd All rights reserved 7
Please cite this article in press as: Tsutakawa et al., Structurally Distinct Ubiquitin- and Sumo-Modified PCNA: Implications for Their Distinct Roles in
the DNA Damage Response, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.02.008
EXPERIMENTAL PROCEDURES
SAXS Analysis of PCNAK164-SUMO and PCNAK107-Ub
SUMOylated and crosslinked yeast PCNA were purified using established protocols (Chen et al., 2010; Freudenthal et al., 2010, 2011). SAXS data were
collected at the SIBYLS 12.3.1 beamline at the Advanced Light Source, Lawrence Berkeley National Laboratory (Classen et al., 2013; Classen et al., 2010;
Hura et al., 2009). Scattering measurements were performed on 20-ml samples
at 15 C (PCNAK164-SUMO) or 22 C (PCNAK107-Ub) loaded into a heliumpurged sample chamber, 1.5 m from the Mar165 detector. Prior to data collection, modified PCNA were purified by size-exclusion chromatography on a
24-ml Superose6 column equilibrated in 20 mM Tris (pH 7.5), 150 mM NaCl,
and 5% glycerol. Data were collected on both the original gel filtration fractions
and samples concentrated 23 from individual fractions (Table S2; Figure S1).
Fractions prior to the void volume and concentrator eluates were used
for buffer subtraction. Sequential exposures (0.5, 0.5, 5, and 0.5 s for
PCNAK164-SUMO and 0.5, 0,5, 2, 5, and 0.5 s for PCNAK107-Ub) were taken
at 12 keV to maximize signal to noise with visual checks for radiation-induced
damage to the protein. Although scattering of the split-fusion PCNAK164SUMO showed radiation-induced aggregation, the scattering of the crosslinked PCNAK107-Ub showed a decrease in slope, indicating that the
molecules in solution were becoming smaller, likely due to irradiation breaking
the disulfide bond. The first and second 0.5-s exposures overlapped, so the
first exposure was assumed to have only minimal damage. The best data,
based on signal-to-noise ratio and Guinier analysis, were collected on splitfusion PCNAK164-SUMO (2.7 mg/ml, 0.5-s exposure) and crosslinked
PCNAK107-Ub (1.1 mg/ml, 0.5-s exposure). Scattering data including c2free
(Rambo and Tainer, 2013) were analyzed using SCATTER available at beamline 12.3.1 (Classen et al., 2013; Rambo and Tainer, 2011). The Porod exponent was determined from a linear regression analysis (I versus q) of the top
of the first peak in the Porod-Debye plot (q4 3 I(q) versus q4) of the scattering
data. P(r) plots were calculated using Gnom implemented in the ATSAS
package.
Computational Models and Protocols
To model the PCNAK107-Ub and PCNAK164-SUMO complexes, we used a
chemically conjugated docking protocol written as a part of the Rosetta 3.4
suite (Baker et al., 2013; Leaver-Fay et al., 2011; Saha et al., 2011). We used
the standard Rosetta scoring function, score12, to rank and select the topscoring models (Kuhlman and Baker, 2000; Rohl et al., 2004). The protocol
was designed to search the conformational space available to ubiquitin
chemically conjugated via an isopeptide bond and sample rotations about
torsional angles in the vicinity of the isopeptide bond. To initiate the sampling
for PCNAK107-Ub, we used the 1UBQ structure for ubiquitin (Vijay-Kumar et al.,
1987) and 1PLQ structure for yeast PCNA (Krishna et al., 1994). For the
PCNAK164-SUMO complex, we used the structure of SUMOylated PCNA
(PDB ID: 3V60) (Armstrong et al., 2012). The initial PDB structures were minimized with the Rosetta relax protocol while using all-heavy-atom constraints
prior to conjugated docking. For the isopeptide linker, protocol UBQ_Gp_
LYX-cterm was used (Baker et al., 2013). Torsions allowed to change included
the c angles of Lys107 or Lys164 of PCNA, the isopeptide bond, and both F
and J angles for the Gly76, Gly75, and Arg74 of ubiquitin (Gly98, Gly97, and
Ile96 of SUMO). Sampling was performed with a standard Rosetta Metropolis
Monte Carlo search protocol (Rohl et al., 2004). Results were automatically
filtered according to the solvent accessible surface area (SASA) buried at
the protein interface (>500 A2) and total score of the docked complex (<0).
In total the sampling produced 4,791 decoys (i.e. Ub/SUMO docking poses)
for PCNA-Ub and 4,499 decoys for PCNA-SUMO using 20,000 Monte Carlo
cycles per trajectory. All Rosetta docking calculations were performed on
the Stampede supercomputer at the Texas Advanced Computing Center
(TACC).
Model Refinement
Outliers with best scores from Rosetta docking were refined using all-atom
explicit solvent MD. In setting up for MD hydrogen atoms, counterions (Na+)
and TIP3P solvent (Jorgensen et al., 1983) were introduced using the XLeap
module in AMBER 12 (Case et al., 2005; Cornell et al., 1995; Duan et al.,
2003). In addition, 100 mM NaCl was introduced to mimic physiological con-
ditions. The systems were then minimized for 5,000 steps with backbone
atoms fixed followed by 5,000 steps of minimization with harmonic restraints
to remove unfavorable contacts. The systems were then gradually brought
up to 300 K and run for 50 ps in the NVT ensemble while keeping the protein
backbone restrained. The equilibration was continued for another 2 ns in
the NPT ensemble and the harmonic restraints were gradually released. The
60-ns production simulations were performed in the NPT ensemble (1 atm
and 300 K) without constraints. A cutoff of 10 A was used for the short-range
non-bonded interactions with a switching function at 8.5 A. The long-range
electrostatic interactions were treated with a smooth particle mesh Ewald
method (Essmann et al., 1995). The r-RESPA multiple timestep method (Tuckerman et al., 1992) was adopted with a time step of 2 fs for bonded interactions, 2 fs for short-range non-bonded interactions, and 4 fs for long-range
electrostatic interactions. Bonds between hydrogen atoms and heavy atoms
of the protein were constrained with the SHAKE algorithm. All simulations
were performed with the NAMD 2.8 code (Kale et al., 1999; Phillips et al.,
2005) using the AMBER Parm99SB force field. Models that departed substantially from the initial Rosetta docking position during MD were eliminated from
further consideration.
MES
Thirteen positions for PCNAK107-Ub (including the 3L10 X-ray structure and
three detached flexible Ub positions identified by averaging from the MD trajectories) and 13 positions for PCNAK164-SUMO (including the 3V60 crystal
structure and three detached flexible SUMO positions) were used to build
triplet structures for the modified complexes. All possible combinations of
positions were generated with three Ub or SUMO moieties per PCNA homotrimer. Thus, we produced a final set of 862 PCNA-Ub triplet models and
1728 PCNA-SUMO triplet models. Computation of scattering profiles from
the models and comparison with the experimental data used the FoXS code
(Schneidman-Duhovny et al., 2010, 2013). The coexistence of different conformations that contribute to the experimental scattering curve had to be taken
into account by considering multiple Ub or SUMO positions. An algorithm
developed by Pelikan et al. (2009) was used to search for the minimal
ensemble of conformations from the pool of all Rosetta-generated triplet
models. The MES included only the minimum set of conformations necessary
to minimize cfree.
SUPPLEMENTAL INFORMATION
Supplemental Information includes two tables and four figures and can be
found with this article online at http://dx.doi.org/10.1016/j.str.2015.02.008.
AUTHOR CONTRIBUTIONS
S.E.T., M.T.W., Z.Z., J.A.T., and I.I. designed research; S.E.T., C.Y., X.X., and
I.I. performed research; B.D.F. and C.P.W., M.T.W., K.Y., and Z.Z. contributed
new reagents/analytic tools; S.E.T., C.Y., X.X., and I.I. analyzed data; and
S.E.T., C.Y., J.A.T, M.T.W., Z.Z., and I.I. wrote the paper.
ACKNOWLEDGMENTS
This work was supported by an NSF CAREER grant MCB-1149521 (to I.I.),
Georgia State University start-up funds (to I.I.), P01 CA092584 (NCI to
J.A.T.), R01 CA081967 (NCI to J.A.T.), NSF Grant MCB-0953764 (to Z.Z.),
and R01 GM108027 (to M.T.W.). Computational resources were provided in
part by a National Science Foundation XSEDE allocation (CHE110042) and
through an allocation at National Energy Research Scientific Computing Center (NERSC) supported by the U.S. Department of Energy Office of Science
(contract DE-AC02-05CH11231). SAXS data were collected at BL12.3.1 at
the Advanced Light Source (ALS), supported by the Integrated Diffraction
Analysis Technologies (IDAT) program (DOE/BER), by DOE contract DEAC02-05CH11231, and by NIH MINOS (R01GM105404).
Received: September 18, 2014
Revised: January 28, 2015
Accepted: February 9, 2015
Published: March 12, 2015
8 Structure 23, 110, April 7, 2015 2015 Elsevier Ltd All rights reserved
Please cite this article in press as: Tsutakawa et al., Structurally Distinct Ubiquitin- and Sumo-Modified PCNA: Implications for Their Distinct Roles in
the DNA Damage Response, Structure (2015), http://dx.doi.org/10.1016/j.str.2015.02.008
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