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Engineering archeal surrogate systems for the development of protein–protein interaction inhibitors against human RAD51

DOI: 10.1016/j.jmb.2016.10.009 DOI Help

Authors: Tommaso Moschetti (University of Cambridge) , Timothy Sharpe (University of Cambridge) , Gerhard Fischer (University of Cambridge) , May E. Marsh (University of Cambridge) , Hong Kin Ng (University of Cambridge) , Matthew Morgan (University of Cambridge) , Duncan E. Scott (University of Cambridge) , Tom L. Blundell (University of Cambridge) , Ashok R. Venkitaraman (Medical Research Council Cancer Unit, University of Cambridge) , John Skidmore (University of Cambridge) , Chris Abell (University of Cambridge) , Marko Hyvonen (University of Cambridge)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Molecular Biology , VOL 428 , PAGES 4589 - 4607

State: Published (Approved)
Published: November 2016
Diamond Proposal Number(s): 315 , 6889 , 7141 , 9007

Open Access Open Access

Abstract: Protein–protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to “humanise” thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from Pyrococcus furiosus. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins.

Journal Keywords: recombinase; protein engineering; humanisation; surrogate system; fragment-based drug discovery

Subject Areas: Medicine, Biology and Bio-materials, Chemistry

Instruments: I02-Macromolecular Crystallography , I03-Macromolecular Crystallography , I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography , I24-Microfocus Macromolecular Crystallography

Other Facilities: Proxima1 at Soleil; ID23-1 at ESRF

Added On: 15/11/2016 09:22


Discipline Tags:

Non-Communicable Diseases Health & Wellbeing Cancer Biochemistry Chemistry Structural biology Drug Discovery Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)