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. 2021 Jan-Jun:296:100773.
doi: 10.1016/j.jbc.2021.100773. Epub 2021 May 11.

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

Aimee J Horsfall  1 , Beth A Vandborg  2 , Wioleta Kowalczyk  3 , Theresa Chav  1 , Denis B Scanlon  1 , Andrew D Abell  4 , John B Bruning  5
Affiliations

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

Aimee J Horsfall et al. J Biol Chem. 2021 Jan-Jun.

Abstract

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 to the features of a PIP-box modulate protein binding and thus how they fine-tune downstream processes. Here, we present a systematic study of each position within the PIP-box to reveal how hPCNA-interacting peptides bind with drastically varied affinities. We synthesized a series of 27 peptides derived from the native protein p21 with small PIP-box modifications and another series of 19 peptides containing PIP-box binding motifs from other proteins. The hPCNA-binding affinity of all peptides, characterized as KD values determined by surface plasmon resonance, spanned a 4000-fold range, from 1.83 nM to 7.59 μM. The hPCNA-bound peptide structures determined by X-ray crystallography and modeled computationally revealed intermolecular and intramolecular interaction networks that correlate with high hPCNA affinity. These data informed rational design of three new PIP-box sequences, testing of which revealed the highest affinity hPCNA-binding partner to date, with a KD value of 1.12 nM, from a peptide with PIP-box QTRITEYF. This work showcases the sequence-specific nuances within the PIP-box that are responsible for high-affinity hPCNA binding, which underpins our understanding of how nature tunes hPCNA affinity to regulate DNA replication and repair processes. In addition, these insights will be useful to future design of hPCNA inhibitors.

Keywords: crystallography; peptides; proliferating cell nuclear antigen (PCNA); structural biology; surface plasmon resonance (SPR).

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Cocrystal structure of p21μ(7KQ1) bound to hPCNA solved by X-ray crystallography. p21μ peptide (purple) shown in cartoon form with side chains as sticks bound to each hPCNA subunit. A, hPCNA shown in cartoon form with transparent surface representation with each subunit indicated in white, gray, and pale purple. B, a hPCNA subunit shown in cartoon ribbons, where the two domains are indicated in green and blue, the IDCL in yellow and the C-terminal tail of hPCNA in orange. C, a zoom-in of a p21μ peptide (purple) bound to hPCNA (gray, surface) where the PIP-box binding site is indicated in pink, the key binding pockets are shown in green, and the conserved PIP-box residues bound are labeled in purple. IDCL, interdomain connecting loop; p21, p21CIP1/WAF1; p21μ, p21 sequence 141 to 155.
Figure 2
Figure 2
Binding affinity (KD) of p21 peptides to PCNA determined by SPR.KD values were calculated using the in-built Biacore Evaluation S200 software and are shown in nanomolar over the respective bar. The bars graphically represent the KD values (in molar, M) as −log(KD[M]). All experiments were repeated to ensure reproducibility, and additional SPR information including fitting errors can be found in Table S2. The dashed line on all panels represents the affinity of p21μ (12.3 nM, −log(KD[M]) = 7.91) to which all modified p21μ peptides are compared throughout the discussion. A, binding affinity of the native p21 peptides. B, rationally mutated p21 peptides with single- or double-point modifications are introduced into the p21 PIP-box. C, binding affinity of peptides containing a native canonical PIP-box from an alternate hPCNA-binding partner (top) or native noncanonical PIP-box from an alternate hPCNA-binding partner (bottom) including two PIP-box sequences which only contain seven amino acids (indicated by the striped bars). The PIP-box sequences are flanked by the same sequence that flanks the p21 PIP-box. D, binding affinity of the rationally designed PIP-box sequences. N.D., could not be determined; p21, p21CIP1/WAF1; SPR, surface plasmon resonance.
Figure 3
Figure 3
Cocrystal structures of p21μ(7KQ1, purple) and p21μ–F150Y (7KQ0, blue) bound to hPCNA solved by X-ray crystallography, compared with p21139–160(1AXC, yellow).A, overall binding mode and structure of p21 peptides bound to hPCNA is the same. Peptide and hPCNA shown are cartoons. B, an overlay of peptide structures shows the PIP-box residues (labeled) adopt similar orientations. C and D, intramolecular (yellow dashed lines) and intermolecular (red dashed lines) polar interactions of p21μ (purple, C) and p21μ–F150Y (blue, D) shown as sticks bound to hPCNA in gray. Conserved PIP-box residues, and the N-terminal residue, are labeled in the corresponding peptide color. p21, p21CIP1/WAF1; p21μ, p21 sequence 141 to 155.
Figure 4
Figure 4
Superimpositionof cocrystallized or computationally modeled peptide:hPCNA structures. Canonical PIP-box peptides are shown in purple, and noncanonical PIP-box peptide structures are shown in green. Canonical: p21μ, p21μ–F150Y p21μ–S146R, p21μ–M147I, p21μ–D149E, p21μ–FY150/151YF, p21μ–Pogo, and p21μ–pol δp66. Noncanonical: p21μ–pol ι, p21μ–PARG, and p21μ–RFC. A, all peptides adopt a single 310-helical turn upon binding hPCNA. B, conserved PIP-box residues shown with sticks show a high degree of similarity. C, flanking residues shown with sticks are orientated in similar directions but are still flexible. D, nonconserved PIP-box residues shown with sticks and adopt a large variety of orientations. p21, p21CIP1/WAF1; p21μ, p21 sequence 141 to 155.
Figure 5
Figure 5
Representative examples of key intermolecular and intramolecular interactions. Structures shown in cartoon format with side chains as sticks. p21μ structure shown in the center in black and the area of key interactions shown in colored rectangles. hPCNA is shown in gray. Computationally modeled peptides bound to hPCNA: p21μ–pol ι, light green; p21μ–S146R, yellow; p21μ–M147I, orange; p21μ–pol δp66, purple; p21μ–PARG, red; p21μ–D149E, light blue; p21μ–Pogo, dark blue; and p21μ–FY150/151YF, dark green. Cocrystal structures of peptides bound to hPCNA: p21μ–F150Y, pink and p21μ, purple. Intramolecular hydrogen bonds are indicated as a yellow dashed line, and intermolecular hydrogen bonds are indicated as a red dashed line. Distances are indicated in angstroms. Elemental coloring: nitrogen, blue; oxygen, red; sulfur, yellow. p21, p21CIP1/WAF1; p21μ, p21 sequence 141 to 155.
Figure 6
Figure 6
Summary of the guideline to design a high-affinity PIP-box and the interactions each residue participates in when bound to hPCNA. hPCNA residues shown as line structures. Intermolecular interactions to side chain or main chain shown as black dashed lines, intramolecular interactions from side chain or main chain shown as red dashed lines.
Figure 7
Figure 7
Computational modeling of rationally designed PIP-box peptidesp21μ–RD1, p21μ–RD2, and p21μ–RD3 on hPCNA. hPCNA shown in gray as a cartoon, with interacting amino acids shown as sticks and labeled in gray. Intermolecular polar interactions are shown as red dashed lines and intramolecular polar interactions shown as yellow dashed lines. A, overlay of the structure of the rationally designed PIP-box peptides. B, p21μ–RD1 shown in orange and conserved residues labeled. C, p21μ–RD2 shown in green and conserved residues labeled. D, p21μ–RD3 shown in pink and conserved residues labeled. p21μ, p21 sequence 141 to 155; p21, p21CIP1/WAF1.
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