Biophysical Insights into a Cryptic Ligand Site in the Hydrophobic Core of Human PCNA.

Publication/Presentation Date

5-20-2026

Abstract

Proteins contain small pockets that form due to imperfections in residue packing or the rotational and conformational movement of amino acids. In this work, we used the fluorescent probe 1-aminoanthracene (AMA) to detect a small ligand pocket in the hydrophobic core of human proliferating cell nuclear antigen (PCNA), which is a critical protein for DNA replication and repair. Fluorescence measurements of AMA reported that the core of PCNA had a dielectric constant (ε) of 4, which was very apolar and similar to cyclohexane (ε = 2). Protein mutagenesis, photoaffinity labeling, and molecular dynamics simulations localized the binding site for AMA next to PCNA residues L90 and L101, which also interacted with general anesthetics (sevoflurane and propofol). The ligand binding site was cryptic, i.e., it formed transiently and was only detectable in certain structural states of PCNA. Ligand binding to the cryptic site on PCNA structurally stabilized the trimeric protein and reduced its ability to disassemble and reassemble its subunits. Thus, the cryptic site in PCNA's core serves to destabilize the assembled protein and promotes structural and oligomeric flexibility. Finally, the hydrophobic site is widely conserved among homologous β clamp proteins with a similar fold as PCNA. This work highlights how small fluorescent probes can reveal ligand sites within proteins, defines the chemical features of protein hydrophobic cores, and introduces a novel approach to modulate the oligomeric stability of PCNA.

ISSN

1542-0086

Disciplines

Medicine and Health Sciences

PubMedID

42169412

Department(s)

Fellows and Residents

Document Type

Article

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