Allosteric inhibition of human exonuclease1 (hExo1) through a novel extended β-sheet conformation

• Published in: Biochimica et biophysica acta. General subjects Vol. 1864; no. 12; p. 129730
• Main Authors: Umar, Aminu Argungu, Liddell, Susan, Hussain, Rohanah, Siligardi, Giuliano, Harris, Gemma, Carr, Stephen, Asiani, Karishma, Gowers, Darren M., Odell, Mark, Scott, David J.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2020
• Subjects: 14-3-3 Proteins - metabolism; Allosteric Regulation; Allostery; cancer therapy; Circular dichroism; circular dichroism spectroscopy; DNA; DNA repair; DNA Repair Enzymes - chemistry; DNA Repair Enzymes - metabolism; Enzyme assay; Exodeoxyribonucleases - chemistry; Exodeoxyribonucleases - metabolism; Exonuclease; Humans; Intrinsic disorder; post-translational modification; Proliferating Cell Nuclear Antigen - metabolism; Protein Binding; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; resection; stress response; ultracentrifugation; Intrinsic disorder; Allostery; Exonuclease; Enzyme assay; Circular dichroism
• ISSN: 0304-4165; 1872-8006; 1872-8006
• DOI: 10.1016/j.bbagen.2020.129730

Human Exonuclease1 (hExo1) participates in the resection of DNA double-strand breaks by generating long 3′-single-stranded DNA overhangs, critical for homology-based DNA repair and activation of the ATR-dependent checkpoint. The C-terminal region is essential for modulating the activity of hExo1, containing numerous sites of post-translational modification and binding sites for partner proteins. Analytical Ultracentrifugation (AUC), Dynamic Light Scattering (DLS), Circular Dichroism (CD) spectroscopy and enzymatic assays. AUC and DLS indicates the C-terminal region has a highly extended structure while CD suggest a tendency to adopt a novel left-handed β-sheet structure, together implying the C-terminus may exhibit a transient fluctuating structure that could play a role in binding partner proteins known to regulate the activity of hExo1. Interaction with 14–3-3 protein has a cooperative inhibitory effect upon DNA resection activity, which indicates an allosteric transition occurs upon binding partner proteins. This study has uncovered that hExo1 consist of a folded N-terminal nuclease domain and a highly extended C-terminal region which is known to interact with partner proteins that regulates the activity of hExo1. A positively cooperative mechanism of binding allows for stringent control of hExo1 activity. Such a transition would coordinate the control of hExo1 by hExo1 regulators and hence allow careful coordination of the process of DNA end resection. The assays presented herein could be readily adapted to rapidly identify and characterise the effects of modulators of the interaction between the 14–3-3 proteins and hExo1. It is conceivable that small molecule modulators of 14–3-3 s-hExo1 interaction may serve as effective chemosensitizers for cancer therapy. •Human Exo1 is predominately an extended monomer in solution.•Human Exo1 C-terminal region forms a relaxed β-sheet conformation.•PCNA and 14–3-3 proteins interact with the C-terminal region of hExo1 to regulate its resection activity.•14–3-3 proteins exhibit positive cooperative inhibition on hExo1.