Abstract
Proliferating cell nuclear antigen (PCNA) is a cellular hub in DNA metabolism and a potential drug target. Its binding partners carry a short linear motif (SLiM) known as the PCNA-interacting protein-box (PIP-box), but sequence-divergent motifs have been reported to bind to the same binding pocket. To investigate how PCNA accommodates motif diversity, we assembled a set of 77 experimentally confirmed PCNA-binding proteins and analyzed features underlying their binding affinity. Combining NMR spectroscopy, affinity measurements and computational analyses, we corroborate that most PCNA-binding motifs reside in intrinsically disordered regions, that structure preformation is unrelated to affinity, and that the sequence-patterns that encode binding affinity extend substantially beyond the boundaries of the PIP-box. Our systematic multidisciplinary approach expands current views on PCNA interactions and reveals that the PIP-box affinity can be modulated over four orders of magnitude by positive charges in the flanking regions. Including the flanking regions as part of the motif is expected to have broad implications, particularly for interpretation of disease-causing mutations and drug-design, targeting DNA-replication and -repair.
Highlights
Protein–protein interactions are essential for all biological processes, especially cellular regulation and signaling
The Proliferating cell nuclear antigen (PCNA) surface is colored in gray shades according to hydrophobicity; the PCNA-interacting proteins (PIPs)-box residues inserted into the binding pockets are highlighted in red and degron specific residues in orange. d Overlay of seven peptides crystallized in complex with human PCNA including a degron (p21) and an AlkB homologue PCNA-interacting motif (APIM) (ZRANB3)
To investigate the primary determinants underlying the affinity between PCNA and its binding partners, we started by considering the effect of secondary structure within the motif region, focusing on the degree of structure preformation and the degree of disorder over the entire length of the motif
Summary
Protein–protein interactions are essential for all biological processes, especially cellular regulation and signaling. The PCNA surface is from the p21-complex (PDB-code: 1AXC) and colored as described in a been estimated to contain more than a hundred thousand— possibly up to a million—different SLiMs, most of which remain to be discovered and understood [4]. This knowledge void limits our understanding of many important biological processes and is rooted in the low sequence conservation of IDPs [5], the existence of only a few core positions of importance in the motif [6, 7], and their experimentally challenging discovery path [8, 9]
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