Defining short linear motif binding determinants by phage-based multiplexed deep mutational scanning

Benz, C.; Maasen, L.; Simonetti, L.; Mihalic, F.; Lindqvist, R.; Tsitsa, I.; Jemth, P.; Overby, A. K.; Davey, N. E.; Ivarsson, Y.

doi:10.1101/2024.08.06.606761

bioRxiv · 10.1101/2024.08.06.606761

Defining short linear motif binding determinants by phage-based multiplexed deep mutational scanning

Benz, C.·Maasen, L.·Simonetti, L.·Mihalic, F.·Lindqvist, R.·Tsitsa, I.·Jemth, P.·Overby, A. K.·Davey, N. E.·Ivarsson, Y.

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Abstract

Deep mutational scanning (DMS) has emerged as a powerful approach for evaluating the effects of mutations on binding or function. Here, we developed a multiplexed DMS by phage display protocol to define the binding determinants of short linear motifs (SLiMs) binding to peptide binding domains. We first designed a benchmarking DMS library to evaluate the performance of the approach on well-known ligands for eleven different peptide binding domains, including the talin-1 PTB domain. Systematic benchmarking against a gold-standard set of motifs from the eukaryotic linear motif (ELM) database confirmed that the DMS by phage analysis correctly identifies known motif binding determinants. The DMS analysis further defined a non-canonical PTB binding motif, with a putative extended conformation. A second DMS library was designed aiming to provide information on the binding determinants for 19 SLiM-based interactions between human and SARS-CoV-2 proteins. The analysis confirmed the affinity determining residues of viral peptides binding to host proteins, and refined the consensus motifs in human peptides binding to five domains from SARS-CoV-2 proteins, including the non-structural protein (NSP) 9. The DMS analysis further pinpointed mutations that increased the affinity of ligands for NSP3 and NSP9. An affinity improved cell-permeable NSP9-binding peptide was found to exert stronger antiviral effects as compared to the initial wild-type peptide. Our study demonstrates that DMS by phage display can efficiently be multiplexed and applied to refine binding determinants, and shows how DMS by phage display can guide peptide-engineering efforts.

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Benz, C., Maasen, L., Simonetti, L., Mihalic, F., Lindqvist, R., Tsitsa, I., Jemth, P., Overby, A. K., Davey, N. E., Ivarsson, Y.. 2024-08-06. Defining short linear motif binding determinants by phage-based multiplexed deep mutational scanning. https://doi.org/10.1101/2024.08.06.606761

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