Abstract
The expanding roles of PCNA in functional assembly of DNA replication and repair complexes motivated investigation of the structural and dynamic properties guiding specificity of PCNA-protein interactions. A series of biochemical and computational analyses were combined to evaluate the PIP Box recognition features impacting complex formation. The results indicate subtle differences in topological and molecular descriptors distinguishing both affinity and stoichiometry of binding among PCNA-peptide complexes through cooperative effects. These features were validated using peptide mimics of p85α and Akt, two previously unreported PCNA binding partners. This study characterizes for the first time a reverse PIP Box interaction with PCNA. Small molecule ligand binding at the PIP Box interaction site confirmed the adaptive nature of the protein in dictating overall shape and implicates allosterism in transmitting biological effects.
Highlights
PCNA has emerged as an essential protein to promote formation of numerous protein complexes in order to regulate cellular processes associated with a DNA damage response [1,2,3]
POGO-ligase (PL), a hybrid 16-mer peptide modeled after the PIP Box conserved sequence of DNA ligase and POGO DNA transposase, demonstrated a similar binding affinity to PCNA by isothermal titration calorimetry (ITC) [7]
For self-competition studies, increasing concentrations of the unlabeled POGO ligase (PL) peptide was incubated with 50 nM fluorescein isothiocyanate (FITC)-PL peptide and 1 mM
Summary
PCNA has emerged as an essential protein to promote formation of numerous protein complexes in order to regulate cellular processes associated with a DNA damage response [1,2,3]. The traditional view of PCNA as a simple processivity factor is being replaced by increasing knowledge of the context-dependency of PCNA complexes that implicate changes in structural features to accommodate functional properties. While the structural scaffold concept holds true for understanding how these proteins interact with PCNA, regulation of the complexes depends on the conformation of the binding partner(s) and potentially on the conformation of PCNA itself. Initial studies investigating how proteins interact with PCNA have identified a variety of conserved sequence motifs and topological relationships that select among these interactions. Mutagenesis and structural data identified a PCNA-protein interaction site proximal to the interdomain connecting loop (IDCL) that accommodates the anchoring of the PIP Box, a conserved sequence binding motif [5]. Initial studies that established the importance of the PIP Box interaction with PCNA used a C-terminal peptide modeled after the amino acid sequence of p21. High-resolution structural analyses by X-ray crystallography indicate similarities in the overall binding mode of several PIP Boxes [10,11,12,13]
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