Molecular Basis for Phosphorylation-dependent SUMO Recognition by the DNA Repair Protein RAP80*

Recognition and repair of double-stranded DNA breaks (DSB) involves the targeted recruitment of BRCA tumor suppressors to damage foci through binding of both ubiquitin (Ub) and the Ub-like modifier SUMO. RAP80 is a component of the BRCA1 A complex, and plays a key role in the recruitment process through the binding of Lys63-linked poly-Ub chains by tandem Ub interacting motifs (UIM). RAP80 also contains a SUMO interacting motif (SIM) just upstream of the tandem UIMs that has been shown to specifically bind the SUMO-2 isoform. The RAP80 tandem UIMs and SIM function collectively for optimal recruitment of BRCA1 to DSBs, although the molecular basis of this process is not well understood. Using NMR spectroscopy, we demonstrate that the RAP80 SIM binds SUMO-2, and that both specificity and affinity are enhanced through phosphorylation of the canonical CK2 site within the SIM. The affinity increase results from an enhancement of electrostatic interactions between the phosphoserines of RAP80 and the SIM recognition module within SUMO-2. The NMR structure of the SUMO-2·phospho-RAP80 complex reveals that the molecular basis for SUMO-2 specificity is due to isoform-specific sequence differences in electrostatic SIM recognition modules.

Recognition and repair of double-stranded DNA breaks (DSB) involves the targeted recruitment of BRCA tumor suppressors to damage foci through binding of both ubiquitin (Ub) and the Ub-like modifier SUMO. RAP80 is a component of the BRCA1 A complex, and plays a key role in the recruitment process through the binding of Lys 63 -linked poly-Ub chains by tandem Ub interacting motifs (UIM). RAP80 also contains a SUMO interacting motif (SIM) just upstream of the tandem UIMs that has been shown to specifically bind the SUMO-2 isoform. The RAP80 tandem UIMs and SIM function collectively for optimal recruitment of BRCA1 to DSBs, although the molecular basis of this process is not well understood. Using NMR spectroscopy, we demonstrate that the RAP80 SIM binds SUMO-2, and that both specificity and affinity are enhanced through phosphorylation of the canonical CK2 site within the SIM. The affinity increase results from an enhancement of electrostatic interactions between the phosphoserines of RAP80 and the SIM recognition module within SUMO-2. The NMR structure of the SUMO-2⅐phospho-RAP80 complex reveals that the molecular basis for SUMO-2 specificity is due to isoform-specific sequence differences in electrostatic SIM recognition modules.
The DNA repair process in eukaryotic cells is an indispensible life process responsible for maintaining the fidelity of the genome (1,2). The genomic information encoded within the molecular structure of DNA is relentlessly compromised as a result of factors that include free radicals arising from metabolic processes, radiation, and replication errors (1). By virtue of highly regulated and efficient DNA repair mechanisms, most DNA damage does not progress to viable malignant tumors (1). Among the different kinds of damage that alter DNA structure, double strand breaks are the most deleterious (1). Depending on the nature of DNA damage, checkpoint activation and cell cycle arrest accompany a number of repair pathways, including homologous recombination, non-homologous end joining, or alternative non-homologous end joining repair, which function to combat the damage (3). Similar to many life processes, homologous recombination is governed by the hierarchical and synergistic action of various post-translational modifications, such as phosphorylation, ubiquitination, and SUMOylation (4,5). The severed ends of damaged DNA are sensed by the MRE11/Rad50/NBS1 (MRN) protein complex, followed by recruitment of ATM kinase and its concomitant activation (3,5,6). This results in the phosphorylation of nearby histones, which serves as a marker for initiation of repair (3,5). Phosphorylated histones comprise the binding site for MDC1, which is also phosphorylated by ATM kinase; subsequently, phosphorylated MDC1 recruits the ubiquitination enzyme RNF8, which acts with the Ubc13/Mms2 heterodimer to attach Lys 63 -linked Ub chains at damaged sites, in combination with the Ub 2 ligase RNF168 (3,7,8). One of the biological functions for Lys 63 linked poly-Ub chains is to serve as a signal for BRCA1 recruitment, a key protein that is obligatory for repair of DNA damage, and cell cycle checkpoint activation. RAP80, an 80-kDa nuclear protein, is responsible for recruitment of the BRCA1 A complex (BRCA1, BARD1, BRCC36, Abraxas, and RAP80) to sites of DNA damage by binding Lys 63 -linked Ub chains through tandem ␣-helical UIMs (9,10). In addition to ubiquitination, SUMOylation of different DNA repair proteins by PIAS4, a SUMO-specific E3 ligase, is involved in BRCA1 recruitment (11,12). The well established role for Lys 63 -linked poly-Ub recognition by RAP80 in BRCA1 recruitment was recently modified by the finding that RAP80 possesses a SIM, N-terminal to the tandem UIMs, which is partly responsible for BRCA1 A complex recruitment to DNA damage sites (13,14). Optimal recruitment of BRCA1 to damage sites depends on the combined action of the SIM and UIM of RAP80; this implies that there are two possibilities for SUMO and poly-Ub binding: independent recognition of the individual modifier proteins, or recognition of SUMO-Ub hybrid chains (13,14). Hybrid chain recognition is appealing in comparison to independent modifier binding, as a result of an ϳ80-fold higher affinity for RAP80 as compared with SUMO and Ub binding alone (14). In addition, the requirement of RNF4, a SUMO-binding Ub ligase, for BRCA1 recruitment by RAP80, suggests that hybrid chains are the preferred candidates for RAP80 binding (14).
There are four SUMO isoforms in mammalian cells: SUMO-1, -2, -3, and 4. SUMO-1 shows 45% sequence identity to SUMO-2 and SUMO-3, whereas SUMO-2 and SUMO-3 share 95% sequence identity, and can form poly-SUMO chains. The function of SUMO-4 is currently unknown. Although their sequences and chain forming capabilities vary, all SUMO iso-forms assume a Ub-like fold (15). From a structural perspective, the binding of SUMO to its cognate partners typically involves electrostatic and hydrophobic interactions, unlike Ub interactions, which typically involve a hydrophobic patch centered on Ile 44 (16 -18). The SIM is the most extensively studied SUMO binding motif, with a hydrophobic module (V/I)X(V/I)(V/I), bordered by N-and C-terminal acidic modules (18). The three isoforms of SUMO bind SIMs within a hydrophobic groove between the ␣ 1 helix and the ␤ 2 strand, typically forming an intermolecular ␤-sheet at the interface. The orientation of the ␤ strand has been observed in both parallel and antiparallel conformations depending on the specific SIM sequence and SUMO isoform (19). This is believed to result from the distribution of negatively charged residues adjacent to the hydrophobic SUMO-interacting module from the SIM. This region also possesses serine residues that are typically phosphorylation sites, and play a role in determining SUMO isoform preference (17,18). Phosphorylation of the SIM serine residues provides enhanced electrostatic interactions, which generally result in a substantial increase in the affinity of the SUMO-SIM interaction (20,21). A number of structures for phosphorylated SIMs bound to SUMO-1 have been reported (19,(21)(22)(23)(24)(25). However, the molecular basis for the interaction between SUMO-2 and its cognate phosphorylated SIM is unknown. The DNA repair protein RAP80 has been shown to preferentially interact with SUMO-2 (14), and possesses a canonical CK2 phosphorylation site within its SIM. In this study, the structure of the N-terminal UIM and SIM domains from RAP80, as well as the molecular basis for binding of SUMO-2 to the SIM were investigated using NMR spectroscopy. We also determined the first structure of SUMO-2 bound to a phosphorylated SIM, which in conjunction with measurement of the thermodynamics and kinetics of SUMO-2 binding for the phosphorylated and non-phosphorylated states of RAP80, provide insight into the molecular determinants underlying the SUMO-2 specificity of this critical DNA repair protein.

Experimental Procedures
Cloning, Protein Expression, and Purification of RAP80-RAP80 is a 719-residue, multidomain protein consisting of N-terminal nuclear localization signals (ϳresidues , two N-terminal tandem UIMs (ϳresidues 80 -120), an N-terminal SIM (ϳresidues [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50], and in the C-terminal half, an Abraxas interacting region and two putative zinc fingers (26). The central Abraxas interacting region binds phosphorylated Abraxas within the BRCA1 complex (9,10,27). This function, combined with the independent SUMO and Ub binding properties of the N-terminal region (ϳresidues 30-120) (13,14,28,29), facilitates DNA damage recognition and repair by the BRCA1 complex. To study the SUMO binding properties of human RAP80, residues 33-131 were cloned into the EcoRI and BamHI sites of pHis-P1, and the insert sequence was verified by sequencing. Expression of the His 6 -tagged fusion construct results in an N-terminal GAMDP cloning artifact following cleavage with tobacco etch virus protease. For expression of unlabeled proteins, 100 l of electrocompetent Escherichia coli strain BL21(DE3)-RIPL cells were transformed with 300 ng of plasmid, and allowed to grow overnight on agar plates containing ampicillin and chloramphenicol at 37°C. A single colony was picked and used to inoculate 50 ml of LB starter culture, which was incubated at 37°C overnight. LB containing ampicillin and chloramphenicol (500 ml) was inoculated with 5 ml of starter culture and incubated at 37°C with shaking at 250 rpm. Upon optimal growth to A 600 ϳ0.6 -0.8, cells were induced with 0.4 mM isopropyl 1-thio-␤-D-galactopyranoside. Post-induction, cells were grown overnight at 25°C and subsequently harvested. Cells were suspended in 100 ml of lysis buffer containing 20 mM imidazole, 500 mM NaCl, 20 mM sodium phosphate, 2 mM DTT, 10 mM MgSO 4 , 5 g/ml of DNase I, and 0.5% protease inhibitor mixture II (Calbiochem catalogue number 538132), pH 7.3, and subjected to sonication. Following cell rupture, lysate was centrifuged at 25,000 rpm in a Beckman JA-25.5 rotor for 30 min at 4°C. The supernatant was filtered using a Millipore steriflip 0.45-m vacuum filtration unit. The filtrate was affinity-purified using a His-prep FF 16/10 column equilibrated with buffer containing 20 mM imidazole, 500 mM NaCl, 20 mM sodium phosphate, and 2 mM DTT, pH 7.3. Bound protein was eluted using a gradient of increasing imidazole concentration ranging from 20 to 500 mM. Fractions containing protein, as detected by UV absorbance, were pooled and the His 6 tag was cleaved by addition of 100 l of 210 M tobacco etch virus protease with incubation at 4°C overnight. The cleaved affinity tag was removed by passing the sample over a His-prep FF 16/10 column; unbound protein was collected and exchanged using a dialysis membrane with a 3.5-kDa cutoff, into buffer containing 50 mM Tris, 150 mM NaCl, and 2 mM DTT, pH 7.3. Final purification was carried out by size exclusion with a HiLoad 26/60 Superdex 75 column equilibrated with 50 mM Tris, 150 mM NaCl, and 2 mM DTT, pH 7.3. Fractions containing protein were pooled and concentrated using an Amicon Ultra 15 centrifugal membrane filtration device with a cutoff of 3 kDa. For expression of [U-15 N]-and [U-13 C, 15 N]-labeled protein for NMR studies, cells were grown to A 600 ϳ0.6 -0.8 in 2 liters of LB media, and pelleted by centrifugation using a Beckman JA-10.5 rotor for 30 min at 5,000 rpm. Cells were washed in M9 medium, and suspended in 250 ml of M9 media containing 15 N-labeled ammonium sulfate as the sole nitrogen source or both 15 N-labeled ammonium sulfate and 13 C-labeled glucose as the sole nitrogen and carbon sources, respectively. Cells were acclimatized to the change in media conditions for ϳ2 h through incubation at 25°C with shaking at 250 rpm. Protein expression was induced using 0.4 mM isopropyl 1-thio-␤-D-galactopyranoside. Following induction, the incubation temperature was reduced to 18°C for overnight growth. Purification was achieved as described for unlabeled proteins.
Protein Expression and Purification of SUMO-2-pET28a plasmid harboring residues 1-93 of human SUMO-2 was a gift from Dr. Lawrence McIntosh, University of British Columbia. Expressed SUMO-2 contained an N-terminal His 6 tag, which results in a GSH cloning artifact following cleavage by thrombin. Protein expression and purification strategies for unlabeled, [U-15 N]-and [U-13 C, 15 N]-labeled SUMO-2 were similar to those for RAP80-(33-131), except where noted. Thrombin cleavage of the SUMO-2 His 6 tag was carried out using a Thrombin CleanCleave kit (Sigma). Cleaved SUMO-2 was dia-lyzed against cleavage buffer containing 50 mM Tris-HCl and 2.5 mM CaCl 2 , pH 7.9, followed by incubation with thrombinimmobilized agarose beads at room temperature for 48 h. Postcleavage, thrombin beads were removed by centrifugation and the protein was dialyzed in nickel column binding buffer (20 mM imidazole, 20 mM sodium phosphate, 500 mM NaCl, 2 mM DTT, pH 7.3). Purification by size exclusion chromatography was similar to that for RAP80- . For all protein samples, purity and molecular weight were confirmed using SDS-PAGE and MALDI-TOF mass spectrometry.
For structure calculations, the starting model for SUMO-2 was derived from the high-resolution crystal structure (PDB code 1WM2) (52). N terminally acetylated and C terminally amidated pRAP80- (37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49) peptide was manually docked to strand ␤ 2 from SUMO-2, in the parallel ␤-strand conformation, to generate a starting model for the complex. The choice for this initial model was based on the following experimental observations, which suggested a parallel ␤-strand conformation for peptide: intermolecular NOEs between SUMO-2 and RAP80 residues Ile 41 /Ile 43 , combined with a lack of intermolecular NOEs between Phe 40 and Val 42 from RAP80 and SUMO-2, significant main chain amide chemical shift perturbations for a network of positively charged residues including His 17 , His 37 , and Lys 42 for SUMO-2, and corresponding large 1 H ␣ and 1 H N chemical shift changes for Ser(P) 44 , Asp 45 , and Ser(P) 46 for SIM peptide, upon SUMO-SIM interaction. This initial model was solvated in a truncated octahedral TIP3P water box, with a distance of 24 Å between protein atoms from images in adjacent unit cells. The starting model was energy minimized using the ff14SB force field and the sander program within the Amber 15 suite of biomolecular simulation programs, with pairwise long range electrostatics and van der Waals interactions cut off at 8 Å. In addition, default parameters for phosphoserine bearing a Ϫ2 charge (S2P residue) were employed (53). The simulation system was heated for 50 ps to a temperature of 298 K, solute atoms were subjected to 2 kcal/mol restraints, and allowed to equilibrate to 1 atmosphere pressure for a further 50 ps. The system was then subjected to production dynamics for 40 ps with the inclusion of NMR-derived distance and dihedral restraints. Structural statistics for 20 snapshots from the last 40 ps of the simulations with NMR restraints are given in Table 1.
NMR Lineshape Analyses-For titrations of SUMO-2 with RAP80-(35-50) and pRAP80-(37-49), lineshape analyses were carried out using the Bloch-McConnell equations for two-site chemical exchange, as previously described (54), to yield the kinetics (k on and k off ) of the RAP80 SIM/SUMO-2 interaction, as well as changes in kinetics upon phosphorylation.

Structural Basis for SUMO Binding by Phosphorylated RAP80
dem ␣-helical UIM domains, a specific global-fold is not adopted. Quantitative chemical shift analysis of main chain torsion angles using the TALOS-N program indicates that residues 33-39, N-terminal to the SIM, adopt a random coil conformation (Fig. 1b), and are flexible, as indicated by random coil index derived S 2 values of ϳ0.50 (56). TALOS-N chemical shift analysis indicates that residues 40 -47 that form the SIM, adopt a ␤ strand conformation with a high probability of ϳ0.95 (Fig.  1b). Residues 48 -78 that connect the SIM to the tandem UIMs also adopt a primarily random coil conformation, with higher flexibility, as indicated by an average random coil-derived S 2 value of ϳ0.3. Residues belonging to the tandem UIMs adopt ␣-helical conformations, as previously described (28,29). The LR motif (residues 60 -78), believed to assist in recruitment of BRCA1 to damage sites (57), does not adopt a specific secondary structure and appears flexible.
Chemical Shift Perturbation Mapping for [U-15 N]-RAP80-  upon Interaction with SUMO-2-To delineate the N-terminal residues of RAP80 that interact with SUMO-2, we followed changes in main chain amide resonances for RAP80-(33-131) upon SUMO-2 binding using two-dimensional 1 H-15 N HSQC NMR spectroscopy (Fig. 1a). In general, a significant chemical shift change accompanying a protein-protein interaction can be expected to have a threshold value of ϳ0.1 ppm, up to a maximum of ϳ0.5 ppm (55). There are few residue-specific, significant chemical shift changes for RAP80-(33-131) within the typical range (Fig. 1c). However, the main chain 1 H N and 15 N resonances for residues expected to be directly involved in the interaction with SUMO-2, Phe 40 , Ile 41 , Val 42 , Ile 43 , and Ser 44 , could not be detected upon interaction with SUMO-2 due to extensive line broadening. The resonances for residues adjacent to the hydrophobic SIM residues, on the other hand, Asp 45 , Ser 46 , and Asp 47 , shifted linearly with increasing SUMO-2, without severe line broadening, consistent with fast chemical exchange. These results suggest that RAP80 interacts with SUMO-2 exclusively via the SIM, similar to the interaction of RAP80 with Ub, wherein only UIM residues are directly involved in binding (29).

NMR-monitored Titrations for the [U-15 N]-SUMO-2/ RAP80-(33-131)
Interaction-To gain insight into the molecular basis underlying specificity of the SUMO-2/RAP80 interaction, we employed NMR spectroscopy to determine the RAP80 binding site on SUMO-2 using chemical shift mapping, and to determine a quantitative dissociation constant for the interaction. Two-dimensional 1 H-15 N HSQC NMR spectra of SUMO-2 in the absence and presence of RAP80-(33-131) are shown in Fig. 2a with the per residue combined chemical shift changes shown in Fig. 2b. SUMO-2 main chain amide resonances showing significant changes upon interaction with RAP80-(33-131) are located within strand ␤ 2 , helix ␣ 1 , the loop connecting them, as well as loop residues near the 3 10 helix in free SUMO-2 (Fig. 2c). These regions form the typical SIM interaction site for the various SUMO isoforms (18). Residues Lys 33 and Lys 35 are undetectable as a result of line broadening, and remained unobservable upon saturation of the binding site. Other residues in the binding cleft, including Val 30 (Fig. 2d), Gln 31 , Phe 32 , Ile 34 , Arg 36 , Thr 38 , Leu 40 , Ser 41 , Lys 42 , and Leu 43 , showed linear chemical shift changes that could be followed throughout the titration. The main chain amide resonances for Phe 32 and Leu 40 were broadened at high RAP80-(33-131)/ SUMO-2 ratios. Fitting chemical shift changes for 20 SUMO-2 residues that could be followed during the titration to a 1:1 binding isotherm gives an average dissociation constant (K D ) of 195 Ϯ 33 M. Representative chemical shift changes for Val 30 , and the fit to a 1:1 binding isotherm are shown in Fig. 2, d and (35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)-To facilitate NMR and structural studies, we synthesized peptides encompassing the minimal binding motif from RAP80 (residues 35-50 or 37-49), given the lack of structure adjacent to these regions, and that no significant chemical shift changes are observed beyond these

Structural Basis for SUMO Binding by Phosphorylated RAP80
FEBRUARY 26, 2016 • VOLUME 291 • NUMBER 9 regions in NMR binding studies employing the longer RAP80-(33-131) construct. The synthetic peptides were N terminally amidated and C terminally acetylated to maintain neutrality for the terminal residues, thereby avoiding introduction of nonphysiologically relevant electrostatic interactions. To ensure that the peptides retained the SUMO interactions of the longer RAP80-(33-131) construct, we conducted chemical shift mapping and NMR-monitored titrations to determine the binding site of the RAP80-(35-50) peptide on SUMO-2, as well as the K D and kinetic constants for the interaction, as described in detail below.

Discussion
For RAP80-(33-131), two-dimensional 1 H-15 N NMR spectra, and quantitative chemical shift analyses were used to derive per residue flexibility and main chain secondary structure, and indicate that the N-terminal region of RAP80 consists of three independent domains. These domains include a partially struc-tured SIM, and two partially structured, tandem UIMs, surrounded by flexible regions. From chemical shift perturbation mappingandNMR-monitoredtitrationsforvariousRAP80constructs, we observe that the minimal region sufficient for interaction with SUMO-2 encompasses residues 40 -47 from the RAP80 SIM. Similar to Ub binding to the tandem UIMs (29), the RAP80 SIM binds SUMO-2 independently. Furthermore, we determined that residues 50 -78 between the SIM and tandem UIMs are not involved in Ub binding. The affinity of the RAP80/SUMO-2 interaction is weak, with a K D of ϳ200 M, and interestingly, double phosphorylation of the SIM at Ser 44 and Ser 46 gives rise to a ϳ25-fold increase in the affinity of the interaction. NMR lineshape analysis indicates that this is due to a substantial increase in k on for SUMO-2/RAP80 SIM association; a result of enhanced electrostatic interactions between the phosphate groups of Ser 44 /Ser 46 , and a positively charged SUMO-2 region at one end of the SIM interaction site. The key role for electrostatics in the modulation of SUMO/SIM interactions is underscored by a prominent dependence on salt concentration (24). The binding affinity of unphosphorylated RAP80 SIM for SUMO-2 is comparable with the affinities of SIM-C from DAXX for both SUMO-1 and SUMO-2 with K D values of ϳ140 M at 200 mM KCl, but weaker in comparison to the ϳ40 M K D for SIM-N binding to both SUMO-1 and SUMO-2 at 200 mM KCl (24). It should be noted that for these affinity comparisons, SIM-C from DAXX shares the SDSD electrostatic module sequence with RAP80, whereas SIM-N from DAXX does not (DDDD). Upon double phosphorylation of RAP80, the SUMO-2-SIM interaction affinity increases, with a K D ϳ9 M; this is comparable with a K D of ϳ1 M for the tetraphosphorylated PML SIM/SUMO-1 interaction, although the greater affinity for the phospho-PML/SUMO-1 interaction, in comparison to that for RAP80/SUMO-2, is likely a result of lower salt concentration for the former interaction (21,23).
The NMR-derived solution state structure of SUMO-2 in complex with pRAP80-(37-49) is the first structure of SUMO-2 bound to a phosphorylated SIM. The structure of the complex shows a rich variety of electrostatic interactions, as well as key hydrophobic interactions, generally separated into two distinct, but adjacent regions, or modules, of the binding interface, as generally observed for SUMO/SIM interactions. The negatively charged region of RAP80 (Ser(P) 44 -Asp 45 -Ser(P) 46 -Asp 47 ), adjacent to hydrophobic SIM module (Phe 40 -Ile 41 -Val 42 -Ile 43 ), is involved in extensive electrostatic interactions with the positively charged residues of the SIM binding interface on SUMO-2. Specifically, the observed chemical shift changes for the SUMO-2 His 17 and His 37 side chain H ␦2 protons upon phospho-SIM binding, suggest electrostatic interactions with a distance range of 3-5 Å between the histidine N ⑀2 and SIM phosphate oxygen atoms for the histidine-phosphoserine pairs. This is consistent with the known relationship between crystallographic structures and chemical shift changes for histidine side chain H ␦2 protons from RNase A upon interaction with nucleotide phosphate groups (61,62).
Other key electrostatic interactions include the intermolecular hydrogen bonds across the peptide planes from RAP80 and SUMO-2 that form the intermolecular ␤-sheet, as well as intramolecular SIM hydrogen bonds between the phosphoserine side chain ␥-oxygens and their respective main chain amide protons.
The RAP80 SIM-SUMO-2 structure is similar to the mDAXX C-terminal SIM/SUMO-1 interaction (PDB code 4WJP) (21), although there are important differences in the electrostatic module of the binding interface that form the basis of specificity determinants (Fig. 6, e and f). The residues from the SIM hydrophobic modules, FIVI for RAP80 and IIVL for DAXX, insert into the hydrophobic SUMO binding cleft similarly, with the side chain of the second and fourth residues buried, and in direct contact with SUMO. As with other SUMO/ SIM interactions, key structural differences are found in the electrostatic binding module adjacent to the hydrophobic module. Specifically, there are key sequence differences for positively charged residues in the electrostatic SIM binding module of SUMO, which includes a SIM binding loop flanking the interaction site (Fig. 6e, residues Met 40 to Lys 46 , and Arg 36 to Lys 42 in SUMO-1 and SUMO-2, respectively). In the case of the electrostatic phosphorylated SDSD modules from both RAP80 and the C-SIM from DAXX (PDB code 4WJP), the first phosphoserine from RAP80, Ser(P) 44 , forms favorable electrostatic interactions with Lys 35 from strand ␤2 adjacent to the N termi-nus of the binding loop, as well as His 17 from strand ␤1. In contrast, the first phosphoserine from DAXX, interacts with Lys 46 from helix ␣1 of SUMO-1, which lies on the opposite, or C-terminal side of the SIM binding loop. In addition, SUMO-1 lacks an equivalent to the positively charged His 17 SUMO-2 residue at the N-terminal side of the binding loop, and possesses Tyr 21 instead. Furthermore, a key SIM binding loop histidine, His 37 from SUMO-2 and His 43 from SUMO-1, occurs at opposite ends of the SIM binding loop. These differences result in the second phosphate from DAXX interacting with the C-terminal portion of the SIM binding loop, whereas the second phosphate from the RAP80 SIM interacts with the N-terminal portion of the SIM binding loop (Fig. 6f). Thus, the change in orientation for various SIM phosphoserine side chains upon interacting with cognate SUMO isoforms can be attributed to different charge distributions in the electrostatic binding modules from SUMO.
From a broader biological perspective, the N-terminal region from RAP80 connects numerous signaling pathways, including SUMOylation, ubiquitination, phosphorylation, and lysine and acetylation, with DNA damage repair (63,64). For example, it has recently been suggested that phosphorylated SIMs function as a node in a network that connects CK2 signaling with SUMOylation (20). CK2 has also been shown to be involved in double strand break repair through homologous recombination by assisting the association of the DNA repair protein Rad51 to the MRN complex through phosphorylation (63). Double phosphorylation of the canonical CK2 site in RAP80 substantially elevates the binding affinity for SUMO-2, and suggests a deeper role for CK2 in the repair of double-stranded DNA breaks, at least within the context of RAP80-mediated recruitment of BRCA1 to DNA damage sites. Interestingly, the phosphorylation of SIMs may be cross-regulated through acetylation of Lys 33 in SUMO-2 and Lys 37 in SUMO-1, although additional lysines near the SIM binding cleft may be involved (65)(66)(67). From a structural perspective, Lys 33 and Lys 35 are critical for electrostatic interactions with SIM or phosphorylated SIM; their acetylation abolishes SUMO-SIM binding, although not in all cases. In the case of RAP80, it will be of interest to determine whether the interaction with SUMO-2 is regulated by acetylation of Lys 33 and Lys 35 , linking regulation of SUMO acetylation with DNA repair by homologous recombination.
The molecular basis underlying the role of ubiquitination and SUMOylation in the RAP80-mediated function of the DNA damage response is not yet fully understood. It is unclear if these post-translational modifications function independently, or if hybrid SUMO-Ub chain recognition is necessary for RAP80 recruitment to DNA damage sites. The latter model is alluring, given that the poly-SUMO binding Ub ligase RNF4 has been shown to be necessary for BRCA1 recruitment (14), and can catalyze the covalent attachment of Ub to SUMO chains (68). This study provides the first molecular details underlying the SUMO/RAP80 interaction, and its regulation by phosphorylation. It will be of interest to gain a deeper understanding of the interplay between various regulatory and signaling processes in RAP80-mediated BRCA1 recruitment to the DNA damage sites.
Author Contributions-L. S. and Anamika designed the study and wrote the paper. Anamika labeled, expressed, and purified RAP80 and SUMO-2, performed NMR experiments, and analyzed NMR data with assistance from L. S. MD simulations with NMR restraints, and NMR lineshape analyses were conducted and analyzed by L. S. and Anamika. Aromatic HSQC NMR pulse sequences were written by L. S., set up and analyzed by Anamika. All authors analyzed the results and approved the final version of the manuscript.