7M5N

PCNA bound to peptide mimetic with linker

Summary for 7M5N

• Entry DOI: 10.2210/pdb7m5n/pdb
• Descriptor: Proliferating cell nuclear antigen, Peptide mimetic (ACE)RQCSMTCFYHSK(NH2) with linker, 1,3-dimethylbenzene (3 entities in total)
• Functional Keywords: pcna, dna replication, peptide mimetic, replication
• Biological source: Homo sapiens (Human); More
• Total number of polymer chains: 5
• Total formula weight: 89202.79

Authors

Vandborg, B.A.,Bruning, J.B. (deposition date: 2021-03-24, release date: 2021-08-11, Last modification date: 2023-10-18)

Primary citation

Horsfall, A.J.,Vandborg, B.A.,Kikhtyak, Z.,Scanlon, D.B.,Tilley, W.D.,Hickey, T.E.,Bruning, J.B.,Abell, A.D.
A cell permeable bimane-constrained PCNA-interacting peptide.
Rsc Chem Biol, 2:1499-1508, 2021

PubMed Abstract: The human sliding clamp protein known as proliferating cell nuclear antigen (PCNA) orchestrates DNA-replication and -repair and as such is an ideal therapeutic target for proliferative diseases, including cancer. Peptides derived from the human p21 protein bind PCNA with high affinity a 3-helical binding conformation and are known to shut down DNA-replication. Here, we present studies on short analogues of p21 peptides (143-151) conformationally constrained with a covalent linker between , + 4 separated cysteine residues at positions 145 and 149 to access peptidomimetics that target PCNA. The resulting macrocycles bind PCNA with values ranging from 570 nM to 3.86 μM, with the bimane-constrained peptide proving the most potent. Subsequent X-ray crystallography and computational modelling studies of the macrocyclic peptides bound to PCNA indicated only the high-affinity peptide adopted the classical 3-helical binding conformation. This suggests the 3-helical conformation is critical to high affinity PCNA binding, however NMR secondary shift analysis of peptide revealed this secondary structure was not well-defined in solution. Peptide is cell permeable and localised to the cell cytosol of breast cancer cells (MDA-MB-468), revealed by confocal microscopy showing blue fluorescence of the bimane linker. The inherent fluorescence of the bimane moiety present in peptide allowed it to be directly imaged in the cell uptake assay, without attachment of an auxiliary fluorescent tag. This highlights a significant benefit of using a bimane constraint to access conformationally constrained macrocyclic peptides. This study identifies a small peptidomimetic that binds PCNA with higher affinity than previous reported p21 macrocycles, and is cell permeable, providing a significant advance toward development of a PCNA inhibitor for therapeutic applications.

PubMed: 34704055
DOI: 10.1039/d1cb00113b

Experimental method

X-RAY DIFFRACTION (3.11 Å)