Abstract
PCNA is an essential factor for DNA replication and repair. It forms a ring shaped structure of 86 kDa by the symmetric association of three identical protomers. The ring encircles the DNA and acts as a docking platform for other proteins, most of them containing the PCNA Interaction Protein sequence (PIP-box). We have used NMR to characterize the interactions of PCNA with several other proteins and fragments in solution. The binding of the PIP-box peptide of the cell cycle inhibitor p21 to PCNA is consistent with the crystal structure of the complex. A shorter p21 peptide binds with reduced affinity but retains most of the molecular recognition determinants. However the binding of the corresponding peptide of the tumor suppressor ING1 is extremely weak, indicating that slight deviations from the consensus PIP-box sequence dramatically reduce the affinity for PCNA, in contrast with a proposed less stringent PIP-box sequence requirement. We could not detect any binding between PCNA and the MCL-1 or the CDK2 protein, reported to interact with PCNA in biochemical assays. This suggests that they do not bind directly to PCNA, or they do but very weakly, with additional unidentified factors stabilizing the interactions in the cell. Backbone dynamics measurements show three PCNA regions with high relative flexibility, including the interdomain connector loop (IDCL) and the C-terminus, both of them involved in the interaction with the PIP-box. Our work provides the basis for high resolution studies of direct ligand binding to PCNA in solution.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Cyclin-Dependent Kinase 2 / metabolism
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Cyclin-Dependent Kinase Inhibitor p21 / metabolism
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Humans
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Inhibitor of Growth Protein 1
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Intracellular Signaling Peptides and Proteins / metabolism
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Magnetic Resonance Spectroscopy*
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Molecular Sequence Data
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Myeloid Cell Leukemia Sequence 1 Protein
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Nuclear Proteins / metabolism
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Peptides / chemistry
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Peptides / metabolism
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Proliferating Cell Nuclear Antigen / chemistry
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Proliferating Cell Nuclear Antigen / metabolism*
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Protein Binding
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Protein Structure, Secondary
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Protein Structure, Tertiary
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Proto-Oncogene Proteins c-bcl-2 / metabolism
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Solutions
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Thermodynamics
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Tumor Suppressor Proteins / metabolism
Substances
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Cyclin-Dependent Kinase Inhibitor p21
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ING1 protein, human
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Inhibitor of Growth Protein 1
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Intracellular Signaling Peptides and Proteins
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Myeloid Cell Leukemia Sequence 1 Protein
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Nuclear Proteins
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Peptides
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Proliferating Cell Nuclear Antigen
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Proto-Oncogene Proteins c-bcl-2
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Solutions
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Tumor Suppressor Proteins
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Cyclin-Dependent Kinase 2
Grants and funding
This work was supported by the Spanish Ministry of Economic Affairs and Competitiveness (
www.mineco.gob.es)grants CTQ2011-28680 to FJB and BIO2009-13265-CO2-01 to IL, and by the grant BIPEDD-CM (P-BIO-0214-2006) from Comunidad Autónoma de Madrid (
www.madrid.org) to RCO. ADB was supported by a Juan de la Cierva contract from the Spanish Ministry of Economic Affairs and Competitiveness. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.