Unlocking the Secrets of NSP3: AlphaFold2-assisted Domain Determination in SARS-CoV-2 Protein

Non-structural protein 3 (nsp3) is crucial for the SARS-CoV-2 infection cycle. It is the largest protein of the virus, consisting of roughly 2000 residues, and a major drug target. However, due to its size, disordered regions, and transmembrane domains, the atomic structure of the whole protein has not yet been established. Only 10 out of its 16 domains were individually determined in experiments. Here, we demonstrate how structural bioinformatics, AI-based fold prediction, and traditional experiments complement each other and can shed light on the makeup of this important protein, both in SARS-CoV-2 and related viruses. Our method can be generalized for other multi-domain proteins, so we describe it in detail. Our prediction-based approach reveals a previously undescribed folded domain, which we could confirm experimentally. Our research also suggests a potential function of the nidovirus-wide conserved domain Y1: This domain may be involved in the assembly of nsp3, nsp4, and nsp6 into the hexameric pore, which was discovered by electron tomography and exports RNA into the cytosol. The Y1-hexamer, however, could not be expressed and purified on its own. We also provide a revised domain segmentation and nomenclature of nsp3 domains based on a compilation of previous research and our own findings.