Unlocking the PIP-box: A peptide library reveals interactions that drive high-affinity binding to human PCNA

• DOI: 10.1016/j.jbc.2021.100773
• Publication Date: 2021-05-11T07:35:11Z
• Authors: Aimee J. Horsfall; Beth A. Vandborg; Wioleta Kowalczyk; Theresa Chav; Denis B. Scanlon; Andrew D. Abell; John B. Bruning
• Keywords: Rational design

The human sliding clamp, Proliferating Cell Nuclear Antigen (hPCNA), interacts with over 200 proteins through a conserved binding motif, the PIP-box, to orchestrate DNA replication and repair. It is not clear how changes features of PIP-box modulate protein thus they fine-tune downstream processes. Here, we present systematic study each position within reveal hPCNA-interacting peptides bind drastically varied affinities. We synthesized series 27 derived from native p21 small modifications another 19 containing motifs other proteins. hPCNA-binding affinity all peptides, characterized as KD values determined by surface plasmon resonance, spanned 4000-fold range, 1.83 nM 7.59 μM. hPCNA-bound peptide structures X-ray crystallography modeled computationally revealed intermolecular intramolecular interaction networks that correlate high hPCNA affinity. These data informed rational design three new sequences, testing which highest partner date, value 1.12 nM, QTRITEYF. This work showcases sequence-specific nuances are responsible for high-affinity binding, underpins our understanding nature tunes regulate repair In addition, these insights will be useful future inhibitors. Human (hPCNA) member clamp family acts an essential processivity factor mediator (1De Biasio A. Blanco F.J. cell nuclear antigen structure interactions: Too many partners one dancer?.Adv. Protein Chem. Struct. Biol. 2013; 91: 1-36Crossref PubMed Scopus (53) Google Scholar, 2Tsurimoto T. PCNA proteins.Front. Biosci. 1999; 4: D849-D858Crossref 3Maga G. Hubscher U. (PCNA): A dancer partners.J. Sci. 2003; 116: 3051-3060Crossref (829) 4Moldovan G.L. Pfander B. Jentsch S. PCNA, maestro fork.Cell. 2007; 129: 665-679Abstract Full Text PDF (1252) 5Boehm E.M. Gildenberg M.S. Washington M.T. roles in eukaryotic replication.Enzymes. 2016; 39: 231-254Crossref (126) Scholar). upregulated majority cancers cope increased demand replication. KO lethal (6Stoimenov I. 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Mol. 2004; 78: 227-260Crossref (237) More than known interact during replication, repair, cell-cycle regulation. However, factors control regulation recruitment appropriate partner, at correct time location, well understood. others have suggested large portion this arises differences hPCNA, span four orders magnitude (3Maga 10Horsfall A.J. Abell A.D. Bruning J.B. mimetics therapeutic purposes.ChemBioChem. 2019; 21: 442-450Crossref (9) 11Prestel Wichmann Martins J.M. Marabini Kassem Broendum S.S. Otterlei Nielsen O. Willemoes Ploug Boomsma W. Kragelund B.B. revisited: Thinking outside PIP-box.Cell. Life 76: 4923-4943Crossref (28) 12Gonzalez-Magana structure, function interactions.Biomolecules. 2020; 10: 570Crossref (21) Such compete gain access where successful ultimately dictated regulator protein, p21CIP1/WAF1 (referred herein p21), (PIP) (KD 2.5–90 (13Wegener K.L. McGrath A.E. Dixon N.E. Oakley Scanlon D.B. Rational 310-helical mimetic targeting - clamp.Chem. Eur. 2018; 24: 11325-11331Crossref 14Bruning Shamoo Y. thermodynamic analysis polymerase-delta p66 flap endonuclease-1.Structure. 12: 2209-2219Abstract (156) 15Gulbis Kelman Hurwitz O'Donnell Kuriyan Structure C-terminal region WAF1/CIP1 complexed PCNA.Cell. 1996; 87: 297-306Abstract (627) Scholar)), upon shuts down consequently stalls progression provide necessary checkpoint healthy proliferation. requires fulfil role outcompete interacting hence fork. contrast, Y-family translesion polymerases pol λ, κ, ι micromolar (16Hishiki Hashimoto H. Hanafusa Kamei Ohashi E. Shimizu Ohmori Sato novel interactions between synthesis proliferating antigen.J. 284: 10552-10560Abstract (107) major processive polymerase (pol δp66 452–466) has 15.6 μM (14Bruning fundamental molecular-level dictate required understand DNA-replication DNA-repair begin unpack subtle sequence influence investigation structure–activity relationship hPCNA. located two domains, nestled under 1C). PIPs (or peptides) consensus aptly named motif. motif defined Qxxϕxxψψ, Q glutamine, 'x' any amino acid, 'ϕ' hydrophobic residue, 'ψ' aromatic residue (commonly Phe or Tyr). Each eight positions referred here 1 8, indicated superscript P1 P8. been argued definition part larger subset noncanonical absence glutamine low nanomolar (11Prestel 17Boehm R.I.P. PIP: PCNA-binding no longer considered specific.Bioessays. 38: 1117-1122Crossref (43) used simplicity discussion. highly canonical p21, 144QTSMTDFY, was initially following alanine mutation scan Lys141 Ser160 Gln144P1, Met147P4, Phe150P7, Tyr151P8 residues (positions 4, 7, 8 (18Warbrick Lane D.P. Glover D.M. Cox L.S. inhibitor defines site cyclin-dependent kinase WAF1 antigen.Curr. 1995; 5: 275-282Abstract (253) 19Warbrick motif.Bioessays. 1998; 20: 195-199Crossref (302) Scholar)). 22-mer peptide, (residues 139–160), contains binds single well-defined turn anchored triad (Met, Phe, Despite insights, scope tolerance number point mutations p21-derived investigated; however, such confined simple functional 20Zheleva D.I. Zhelev N.Z. Fischer P.M. Duff S.V. Warbrick Blake D.G. quantitative vitro domain PCNA: Affinity, stoichiometry, thermodynamics.Biochemistry. 2000; 7388-7397Crossref (67) 21Warbrick sequence, screening design.Oncogene. 2006; 25: 2850-2859Crossref (32) 22Kroker exploits Tyr151 tether binding.Biochemistry. 2015; 54: 3483-3493Crossref For example, Tyr151PheP8 modified demonstrated tyrosine phenol group makes hydrogen bond Gln131 (22Kroker nonconserved 2, 3, 5, 6) p21139–160 only participate stabilizing bonds (146–149, 147–150, 147–151) (10Horsfall led belief play limited thorough develop nuanced key owes affinity, plays short reported, either one- two-point made determine secondary acids incorporated were chosen reflect those observed respective sequences. second prepared sequences different proteins, inserted flanks sequence. allowed wider array combinations investigated allows direct comparison effect contain same flanking PIP-box. resonance (SPR). bound studied computational modeling studies, correlated uncover studies inform investigate whether cooperative could predicted peptides. comprehensive advances ability predict it provides insight into fine-tuned can leveraged further regulates process inhibitors interactions. Fifty-one solid-phase (see Experimental procedures) allow (Table S1). comprise hPCNA; point-modified peptides; included PIP-box; final set entirely new, rationally designed 141 155 p21μ) reported shortened without impacting Interestingly, preparation shorter gave less aspartimide formation compared 22mer, M-18 peak mass spectrum apparent p21μ, contrast (unpublished work), thereby improving synthetic yields. All subsequent based p21μ 141–155). Five 144P1 prepared: p21μ–Q144K, p21μ–Q144M, p21μ–Q144D, p21μ–Q144S, p21μ–Q144N. Four inspired residues: lysine ι, κ), methionine (Cdt2, η RNaseH2B), aspartic acid (poly(ADP-ribose) glycohydrolase, PARG), serine λ) 1. asparagine amidated, but side chain (p21μ–Q144N). amino-acid modification 4 include valine (p21μ–M147V), isoleucine (p21μ–M147I), leucine (p21μ–M147L), commonly smaller still (p21μ–M147A) nonpolar tryptophan (p21μ–M147W). Aromatic residues, phenylalanine tyrosine, 7 PheP7TyrP8 combination permutation (p21μ–Y151F, p21μ–F150Y, p21μ–FY150/151YF). p21μ–F150H prepared, PARG Positively charged 2 3 (e.g., XPG, FEN1, WRN, PARG, p15, Cdt1). Consequently, arginine and/or (p21μ–T145K, p21μ–T145R, p21μ–S146K, p21μ–S146R, p21μ–TS145/146KK, p21μ–TS145/146KR, p21μ–TS145/146RK, p21μ–TS145/146RR). Conversely, negatively 5 6 Cdt1, RNaseH2B) five glutamic (p21μ–T148D, p21μ–T148E, p21μ–TD148/149DE, p21μ–TD148/149EE, p21μ–D149E). Finally, variety combinations, replaced peptide. Ten Pogo, DNALig1, MCMT, δp66, RecQ5 chosen, along Cdt2, η, RNaseH2B, RFCp14. terminus does extending C- N-terminal δp66). influenced (KD) SPR, results summarized Figure Table S2. representative sample SPR sensorgrams Fig. S1. p21139–160, positive control, 4.32 2A, S2), agrees previous literature 141–155) 12.3 indicates truncated tolerated, although seven S2). throughout discussion, unless otherwise indicated. marked dashed line panels benchmark comparison. magnitude, best having (p21μ–TS145/146RR) lowest 8.14 (p21μ–pol B C, p21μ–Cdt2 p21μ–RecQ5 because nonspecific sensor chip. higher p21μ: p21μ–TS145/146RR (1.83 nM), p21μ–S146R (4.30 p21μ–Cdt1 (8.76 p21μ–Pogo (8.82 p21μ–Y151F (10.6 p21μ–M147I (11.1 nM) C). (with Gln144P1) range 3.57 2C, 'canonical'). general, group, unsurprisingly, lower 2C). conformation potential reasons Cocrystal (PDB ID: 7KQ1, 3.30 Å) p21μ–F150Y 7KQ0, 2.40 solved. Both served important controls studies: showed similar manner (position structure) 22mer 1AXC), proof concept represented crystal accurately. resulting shown Figs. S1 S2, collection refinement statistics S3. cocrystal 1) six 3C, S2 S4). 144 151 retain conformations (1AXC, B), RMSD 0.51 Å S18). also reflected similarity buried area 1AXC S19). Gln144P1 Ala252 Pro253 Q-pocket, Ala208, formed bonds. Met147P4 packed slightly differently analogous (1AXC). (Lys254-Asp257) (141–143) electron density, (1AXC) electrostatic Arg142 Glu256 Asp257 3C). After this, selected high-throughput more structures. 7KQ1) starting most overall 1AXC). p21μ–M147I, p21μ–D149E, p21μ–FY150/151YF resulted largest increase position. hPCNA: p21μ–pol δp66; p21μ–PARG, p21μ–RFC (Figs. S2–S15). 4A), average 0.223 suggests difference due structural changes. adopted 4B); much C D). Intramolecular (peptide-protein) (peptide-peptide) Tables S4, S5–S17. co-crystal influences conformation. (15Gulbis 452–466 (1U76, ι415–437 (2ZVM, PARG402–420 (5MAV, (23Kaufmann Grishkovskaya Polyansky A.A. Kostrhon Kukolj Olek K.M. Herbert Beltzung Mechtler Peterbauer Gotzmann Zhang L. Hartl Zagrovic Elsayad et al.A non-canonical mediates PCNA.Nucleic Acids 45: 9741-9759Crossref (26) Pogo mutant Pogo-Ligase (1VYJ, (24Kontopidis Wu S.-Y. Zheleva Taylor P. McInnes C. Walkinshaw M.D. biochemical complexes rationale cyclin design.Proc. Natl. Acad. 2005; 102: 1871-1876Crossref (103) overlaid S16), (RMSD S20) S21). adopt counterpart 0.51, 0.69, 0.49, 0.69 Å, respectively S20, S16). PIP-box–containing very conformations, 1.13 S16 S20). information harnessed attempt test requirements binding. mimic favorable 5). particular primary mind appeared stabilize highlighted then scaffold, before, give p21μ–RD1–3. 2D) reve