A quantitative study of the in vitro binding of the C-terminal domain of p21 to PCNA

affinity, stoichiometry, and thermodynamics

Daniella I. Zheleva*, Nikolai Z. Zhelev, Peter M. Fischer, Susan V. Duff, Emma Warbrick, David G. Blake, David P. Lane

*Corresponding author for this work

Research output: Contribution to journalArticle

57 Citations (Scopus)

Abstract

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and control of cell proliferation, and its activity can be modulated by interaction with p21(Wafl/Cip1) [Cox, L. S., (1997) Trends Cell Biol. 7, 493-497]. This protein-protein interaction provides a particularly good model target for designing therapeutic agents to treat proliferative disorders such as cancer. In this study, the formation of complexes between PCNA and peptides derived from the C-terminus of p21 has been investigated at the molecular level and quantified using a competitive PCNA binding assay and isothermal titration calorimetry (ITC). The affinity constant for the interaction between p21 (141-160) peptide and PCNA has been determined to be 1.14 x 107 M-1, corresponding to a Kd 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 141-152) of the above peptide is the minimum recognition motif, required for PCNA binding. Truncation of the C-terminal region p21 (153160), though, inhibited significantly the ability of the peptides to compete with the full-length p21 (141160) for binding to PCNA. Alanine mutation of Met 147 or Asp 149 completely abolished or significantly decreased, respectively, the level of the PCNA binding and the inhibition of SV40 DNA replication. Comparison of the data obtained by the competitive PCNA binding assay and the ITC measurements demonstrated the usefulness of this assay for screening for compounds that could modulate the PCNAp21 interaction. Using this assay, we have screened rationally designed peptides for binding to PCNA and interruption of the PCNA-p21 (141-160) complex. As a result of this screening, we have identified a 16-residue peptide (consensus motif 1 peptide) with the following sequence: SAVLQKKITDYFHPKK. Consensus motif 1 peptide and p21 (141-160) have similar affinities for binding PCNA and abilities to inhibit in vitro replication of DNA originated from SV40. Such peptides could prove useful in assessing p21-mimetic strategies for cancer treatment.

Original languageEnglish
Pages (from-to)7388-7397
Number of pages10
JournalBiochemistry
Volume39
Issue number25
Early online date27 May 2000
DOIs
Publication statusPublished - 1 Jun 2000
Externally publishedYes

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Proliferating Cell Nuclear Antigen
Thermodynamics
Stoichiometry
Peptides
Assays
DNA Replication
Calorimetry
Titration
In Vitro Techniques
DNA
Screening
Oncology
Cell proliferation
DNA Repair
Alanine
Neoplasms
Proteins
Repair
Substitution reactions
Cell Proliferation

Cite this

Zheleva, D. I., Zhelev, N. Z., Fischer, P. M., Duff, S. V., Warbrick, E., Blake, D. G., & Lane, D. P. (2000). A quantitative study of the in vitro binding of the C-terminal domain of p21 to PCNA: affinity, stoichiometry, and thermodynamics. Biochemistry, 39(25), 7388-7397. https://doi.org/10.1021/bi992498r
Zheleva, Daniella I. ; Zhelev, Nikolai Z. ; Fischer, Peter M. ; Duff, Susan V. ; Warbrick, Emma ; Blake, David G. ; Lane, David P. / A quantitative study of the in vitro binding of the C-terminal domain of p21 to PCNA : affinity, stoichiometry, and thermodynamics. In: Biochemistry. 2000 ; Vol. 39, No. 25. pp. 7388-7397.
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abstract = "Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and control of cell proliferation, and its activity can be modulated by interaction with p21(Wafl/Cip1) [Cox, L. S., (1997) Trends Cell Biol. 7, 493-497]. This protein-protein interaction provides a particularly good model target for designing therapeutic agents to treat proliferative disorders such as cancer. In this study, the formation of complexes between PCNA and peptides derived from the C-terminus of p21 has been investigated at the molecular level and quantified using a competitive PCNA binding assay and isothermal titration calorimetry (ITC). The affinity constant for the interaction between p21 (141-160) peptide and PCNA has been determined to be 1.14 x 107 M-1, corresponding to a Kd 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 141-152) of the above peptide is the minimum recognition motif, required for PCNA binding. Truncation of the C-terminal region p21 (153160), though, inhibited significantly the ability of the peptides to compete with the full-length p21 (141160) for binding to PCNA. Alanine mutation of Met 147 or Asp 149 completely abolished or significantly decreased, respectively, the level of the PCNA binding and the inhibition of SV40 DNA replication. Comparison of the data obtained by the competitive PCNA binding assay and the ITC measurements demonstrated the usefulness of this assay for screening for compounds that could modulate the PCNAp21 interaction. Using this assay, we have screened rationally designed peptides for binding to PCNA and interruption of the PCNA-p21 (141-160) complex. As a result of this screening, we have identified a 16-residue peptide (consensus motif 1 peptide) with the following sequence: SAVLQKKITDYFHPKK. Consensus motif 1 peptide and p21 (141-160) have similar affinities for binding PCNA and abilities to inhibit in vitro replication of DNA originated from SV40. Such peptides could prove useful in assessing p21-mimetic strategies for cancer treatment.",
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Zheleva, DI, Zhelev, NZ, Fischer, PM, Duff, SV, Warbrick, E, Blake, DG & Lane, DP 2000, 'A quantitative study of the in vitro binding of the C-terminal domain of p21 to PCNA: affinity, stoichiometry, and thermodynamics', Biochemistry, vol. 39, no. 25, pp. 7388-7397. https://doi.org/10.1021/bi992498r

A quantitative study of the in vitro binding of the C-terminal domain of p21 to PCNA : affinity, stoichiometry, and thermodynamics. / Zheleva, Daniella I.; Zhelev, Nikolai Z.; Fischer, Peter M.; Duff, Susan V.; Warbrick, Emma; Blake, David G.; Lane, David P.

In: Biochemistry, Vol. 39, No. 25, 01.06.2000, p. 7388-7397.

Research output: Contribution to journalArticle

TY - JOUR

T1 - A quantitative study of the in vitro binding of the C-terminal domain of p21 to PCNA

T2 - affinity, stoichiometry, and thermodynamics

AU - Zheleva, Daniella I.

AU - Zhelev, Nikolai Z.

AU - Fischer, Peter M.

AU - Duff, Susan V.

AU - Warbrick, Emma

AU - Blake, David G.

AU - Lane, David P.

PY - 2000/6/1

Y1 - 2000/6/1

N2 - Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and control of cell proliferation, and its activity can be modulated by interaction with p21(Wafl/Cip1) [Cox, L. S., (1997) Trends Cell Biol. 7, 493-497]. This protein-protein interaction provides a particularly good model target for designing therapeutic agents to treat proliferative disorders such as cancer. In this study, the formation of complexes between PCNA and peptides derived from the C-terminus of p21 has been investigated at the molecular level and quantified using a competitive PCNA binding assay and isothermal titration calorimetry (ITC). The affinity constant for the interaction between p21 (141-160) peptide and PCNA has been determined to be 1.14 x 107 M-1, corresponding to a Kd 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 141-152) of the above peptide is the minimum recognition motif, required for PCNA binding. Truncation of the C-terminal region p21 (153160), though, inhibited significantly the ability of the peptides to compete with the full-length p21 (141160) for binding to PCNA. Alanine mutation of Met 147 or Asp 149 completely abolished or significantly decreased, respectively, the level of the PCNA binding and the inhibition of SV40 DNA replication. Comparison of the data obtained by the competitive PCNA binding assay and the ITC measurements demonstrated the usefulness of this assay for screening for compounds that could modulate the PCNAp21 interaction. Using this assay, we have screened rationally designed peptides for binding to PCNA and interruption of the PCNA-p21 (141-160) complex. As a result of this screening, we have identified a 16-residue peptide (consensus motif 1 peptide) with the following sequence: SAVLQKKITDYFHPKK. Consensus motif 1 peptide and p21 (141-160) have similar affinities for binding PCNA and abilities to inhibit in vitro replication of DNA originated from SV40. Such peptides could prove useful in assessing p21-mimetic strategies for cancer treatment.

AB - Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and control of cell proliferation, and its activity can be modulated by interaction with p21(Wafl/Cip1) [Cox, L. S., (1997) Trends Cell Biol. 7, 493-497]. This protein-protein interaction provides a particularly good model target for designing therapeutic agents to treat proliferative disorders such as cancer. In this study, the formation of complexes between PCNA and peptides derived from the C-terminus of p21 has been investigated at the molecular level and quantified using a competitive PCNA binding assay and isothermal titration calorimetry (ITC). The affinity constant for the interaction between p21 (141-160) peptide and PCNA has been determined to be 1.14 x 107 M-1, corresponding to a Kd 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 141-152) of the above peptide is the minimum recognition motif, required for PCNA binding. Truncation of the C-terminal region p21 (153160), though, inhibited significantly the ability of the peptides to compete with the full-length p21 (141160) for binding to PCNA. Alanine mutation of Met 147 or Asp 149 completely abolished or significantly decreased, respectively, the level of the PCNA binding and the inhibition of SV40 DNA replication. Comparison of the data obtained by the competitive PCNA binding assay and the ITC measurements demonstrated the usefulness of this assay for screening for compounds that could modulate the PCNAp21 interaction. Using this assay, we have screened rationally designed peptides for binding to PCNA and interruption of the PCNA-p21 (141-160) complex. As a result of this screening, we have identified a 16-residue peptide (consensus motif 1 peptide) with the following sequence: SAVLQKKITDYFHPKK. Consensus motif 1 peptide and p21 (141-160) have similar affinities for binding PCNA and abilities to inhibit in vitro replication of DNA originated from SV40. Such peptides could prove useful in assessing p21-mimetic strategies for cancer treatment.

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DO - 10.1021/bi992498r

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JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

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