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Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) of complex multi-domain proteins are now identified as a trend topic by the scientific community. NMR constitutes a unique investigation tool to access atom resolved information on their structural and dynamic properties, in isolation or upon interaction with potential partners (metal ions, small molecules, proteins, nucleic acids, membrane mimetics etc.). Their high flexibility and disorder, in contrast to more compact structures of globular protein domains, has a strong impact on NMR observables and NMR experiments should be tailored for their investigation. In this context, 13C direct detection NMR has become a very useful tool to contribute to IDPs/IDRs characterization at atomic resolution. 2D CON spectra can now be collected in parallel to 2D HN ones, and reveal information, which in some cases is not accessible through 2D HN spectra only, particularly when studying proteins in experimental conditions approaching physiological pH and temperature. The 2D HN/CON spectra are thus becoming a sort of identity card of an IDP/IDR in solution. Their simultaneous acquisition through multiple receiver NMR experiments is particularly useful to investigate the properties of highly flexible intrinsically disordered regions within complex multi-domain proteins, rather than in isolation as often performed to reduce the complexity of the system, an interesting perspective in the field.

Novel and efficient strategies need to be developed to interfere with the SARS-CoV-2 virus. One of the most promising pharmaceutical targets is the nucleocapsid protein (N), responsible for genomic RNA packaging. N is composed of two folded domains and three intrinsically disordered regions (IDRs). The globular RNA binding domain (NTD) and the tethered IDRs are rich in positively charged residues. The study of the interaction of N with polyanions can thus help to elucidate one of the key driving forces responsible for its function, i.e., electrostatics. Heparin, one of the most negatively charged natural polyanions, has been used to contrast serious cases of COVID-19 infection, and we decided to study its interaction with N at the molecular level. We focused on the NTR construct, which comprises the NTD and two flanking IDRs, and on the NTD construct in isolation. We characterized this interaction using different nuclear magnetic resonance approaches and isothermal titration calorimetry. With these tools, we were able to identify an extended surface of NTD involved in the interaction. Moreover, we assessed the importance of the IDRs in increasing the affinity for heparin, highlighting how different tracts of these flexible regions modulate the interaction.

The SARS-CoV-2 nucleocapsid (N) protein is crucial for the highly organized packaging and transcription of the genomic RNA. Studying atomic details of the role of its intrinsically disordered regions (IDRs) in RNA recognition is challenging due to the absence of structure and to the repetitive nature of their primary sequence. IDRs are known to act in concert with the folded domains of N and here we use NMR spectroscopy to identify the priming events of N interacting with a regulatory SARS-CoV-2 RNA element. 13C-detected NMR experiments, acquired simultaneously to 1H detected ones, provide information on the two IDRs flanking the N-terminal RNA binding domain (NTD) within the N-terminal region of the protein (NTR, 1–248). We identify specific tracts of the IDRs that most rapidly sense and engage with RNA, and thus provide an atom-resolved picture of the interplay between the folded and disordered regions of N during RNA interaction.