A quantitative study of the in vitro binding of the C-terminal domain of p21 to PCNA: Affinity, stoichiometry, and thermodynamics

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA re plication, repair, and control of cell proliferation, and its activity can be modulated by interaction with p21(Wa1/Cip1) [Cox, L. S., (1997) Trends C ell Biol. 7, 493-497]. This protein-protein interaction provides a particul arly good model target for designing therapeutic agents to treat proliferat ive disorders such as cancer. In this study, the formation of complexes bet ween PCNA and peptides derived from the C-terminus of p21 has been investig ated at the molecular level and quantified using a competitive PCNA binding assay and isothermal titration calorimetry (ITC). The affinity constant fo r the interaction between p21 (141-160) peptide and PCNA has been determine d to be 1.14 x 10(7) M-1, corresponding to a K-d Of 87.7 nM. Measurement of the interaction of truncation and substitution analogues based on the p21 (141-160) sequence with PCNA revealed that the N-terminal part (residues 14 1-152) of the above peptide is the minimum recognition motif, required for PCNA binding. Truncation of the C-terminal region p21 (153-160), though, in hibited significantly the ability of the peptides to compete with the full- length p21 (141-160) for binding to PCNA. Alanine mutation of Met 147 or As p 149 completely abolished or significantly decreased, respectively, the le vel of the PCNA binding and the inhibition of SV40 DNA replication. Compari son of the data obtained by the competitive PCNA binding assay and the ITC measurements demonstrated the usefulness of this assay for screening for co mpounds that could modulate the PCNA-p21 interaction. Using this assay, we have screened rationally designed peptides for binding to PCNA and interrup tion of the PCNA-p21 (141-160) complex. As a result of this screening, we h ave identified a 16-residue peptide (consensus motif 1 peptide) with the fo llowing sequence: SAVLQKKITDYFHPKK. Consensus motif 1 peptide and p21 (141- 160) have similar affinities for binding PCNA and abilities to inhibit in v itro replication of DNA originated from SV40. Such peptides could prove use ful in assessing p21-mimetic strategies for cancer treatment.