Srs2 possesses a non-canonical PIP box in front of its SBM for precise recognition of SUMOylated PCNA.

Dear Editor, The modification of proliferating cell nuclear antigen (PCNA) by small ubiquitinlike modifier (SUMO) recruits Srs2 during S phase (Hoege et al., 2002; Pfander et al., 2005). The recruitment of Srs2 to replication fork through physical interaction between the C-terminus of Srs2 (residues 1038–1174) and SUMOylated PCNA regulates homologous recombination by disrupting the Rad51 nucleoprotein filament formation, which is an essential step in a recombination pathway (Krejci et al., 2003; Veaute et al., 2003; Pfander et al., 2005; Colavito et al., 2009). Although many studies stressed the importance of the interaction between Srs2 and SUMOylated PCNA in the regulation of homologous recombination, the mechanism of the recognition of SUMOylated PCNA by Srs2 remains unclear at the molecular level. To elucidate the mechanism of recognition of SUMOylated PCNA by Srs2, we first hypothesized that Srs2 might contain a PCNA-interacting motif nearby the SUMObinding motif (SBM; residues 1170–1173; Kerscher, 2007) to distinguish the SUMOylated PCNA from other SUMOylated substrates in the nucleus. To check the existence of a PCNA-interacting motif, we performed pull-down assays using TrxHisSrs2/ 137c (residues 1038–1174; known as the SUMOylated PCNA-binding region; Pfander et al., 2005), TrxHisSrs2/137cDC32 (residues 1038–1142) and TrxHisSrs2/32c (residues 1143–1174), which contain thioredoxin (Trx) and hexahistidine (His) tags at the N-terminus. PCNA co-eluted with TrxHisSrs2/137c and TrxHisSrs2/32c, not with TrxHisSrs2/137cDC32 (Figure 1A). We further acquired sequential heteronuclear single quantum coherence (HSQC) spectra of N-labeled Srs2/32c with various concentrations of unlabeled PCNA. The peaks of Srs2/32c disappeared when unlabeled PCNA was added, and the most perturbed region of Srs2/32c was located N-terminal to the SBM, within residues 1149–1162 (Supplementary Figure S1). To identify the critical residues, we introduced a single alanine mutation at each amino acid position in the most perturbed region of Srs2/32c, based on the nuclear magnetic resonance (NMR) titration result (residues 1149–1162). We observed that mutations at D1151, I1152, F1153, L1156 and K1160 completely abolished PCNA binding, and mutations at M1150, R1158 and K1161 reduced binding affinity significantly (Figure 1B). Notably, the analysis of the critical residues shows that the PCNA-interacting motif of Srs2 does not contain the classical PIP box, and it possesses a hydrophobic core (Figure 1C). Furthermore, it strongly suggested that the basic charged residues at the C-terminus (residues 1159–1162; RAKKK) also play a role in interacting with PCNA, since a single alanine mutation on each positively charged residue shows a significant reduction in binding affinity to PCNA (Figure 1B). These findings demonstrated that Srs2 physically interacts with PCNA through a non-canonical PIP box, which contains the hydrophobic core at the N-terminus and positively charged residues at the C-terminus, similar to the PIP box of p21 (Figure 1C; Gulbis et al., 1996). To examine whether this non-canonical PIP box uses the same binding surface as the classical PIP box, we used an NMR titration. A significant chemical shift perturbation in the hydrophobic pocket and the interdomain connecting loop (IDCL) of PCNA were observed in HSQC spectra of perdeuterated N-labeled PCNA when unlabeled Srs2/32c was added, and this perturbed region is similar to the binding surface on PCNA in the p21–PCNA complex (Figure 1D and Supplementary Figure S2). Additional proof came from a pull-down assay using a PCNA/A251V mutant. The mutant’s bulky side chain is known to disrupt the interaction between the classical PIP box and the hydrophobic pocket in PCNA (Moldovan et al., 2006). In comparison with Figure 1A, no portion of PCNA/A251V mutant co-eluted with TrxHisSrs2/137c or TrxHisSrs2/32c (Supplementary Figure S2). Hence, the non-canonical PIP box in Srs2 shares the same binding surface as the classical PIP box, and the structural nature of the non-canonical PIP box of Srs2 might not differ from the PIP box of p21 in complex with PCNA. We thus used HADDOCK software to generate a structural model of the non-canonical PIP box of Srs2 in complex with yeast PCNA. In the model, the noncanonical PIP box in Srs2 interacts with PCNA across the front side of PCNA in an anti-parallel orientation with respect to the IDCL, like the PIP box in p21 (Figure 1E and Supplementary Figure S2). The hydrophobic core (residue 1148–1153) adopts a 310 helical structure and it anchors on the hydrophobic pocket of PCNA, similar to the PIP box of p21 (Figure 1E). Furthermore, positively charged residues form stable hydrogen bonds with the negatively charged side chains of D120, D122 and D124 on the IDCL. It strongly illustrated that the cluster of basic residues at the C-terminus 258 | Journal of Molecular Cell Biology (2012), 4, 258–261 doi:10.1093/jmcb/mjs026 Published online May 28, 2012