XChem Fragment Screen

Details about the purification and crystallization of NSP13 can be found here. The protein was produced and optimized crystallization and seeding conditions were found in the Gileadi lab at the University of Oxford. Further crystallization, crystal mounting, crystal soaking and X-ray data collection was performed by the XChem team at Diamond Light Source. The fragment screen encompassed the full DSI-poised Library, with an additional small selection of fragments from libraries available at XChem.

The NSP13 crystal form contains two protomers and the fragment screen revealed a total of 63 fragment hits across 51 datasets. These datasets have been submitted to the PDB and are also available to view in fragalysis.

There are several potential sites of inhibition of NSP13 helicase activity, namely the nucleotide and RNA binding sites, and putative allosteric sites that may block inter-domain movements or interactions with protein partners. Structures of NSP13 in complex with nucleotides or nucleic acid substrates are not known but we have modelled in the expected positions based on the Upf-1 RNA complex structure (2XZL) which shares sequence homology with NSP13. Here, the nucleotide binds in a cleft between the 1A and 2A domains, whilst the RNA passes through a cavity formed between the IB domain and the 1A and 2A domains. Our X-ray fragment screen yielded fragment hits in all these sites.

Left: Structural model of NSP13 with the RNA and nucleotide bound in their expected positions based on the crystal structure of Upf-1 RNA complex 2XZL (Chakrabarti et. al. 2011). Right: NSP13 fragment hits viewed from the same orientation.