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Papain-like protease (PLpro) from SARS-CoV-2 plays essential roles in the replication cycle of the virus. In particular, it preferentially interacts with and cleaves human interferon-stimulated gene 15 (hISG15) to suppress the innate immune response of the host. We used small-angle X-ray and neutron scattering combined with computational techniques to study the mechanism of interaction of SARS-CoV-2 PLpro with hISG15. We showed that hISG15 undergoes a transition from an extended to a compact state after binding to PLpro, a conformation that has not been previously observed in complexes of SARS-CoV-2 PLpro with ISG15 from other species. Furthermore, computational analysis showed significant conformational flexibility in the ISG15 N-terminal domain, suggesting that it is weakly bound to PLpro and supports a binding mechanism that is dominated by the C-terminal ISG15 domain. This study fundamentally improves our understanding of the SARS-CoV-2 deISGylation complex that will help guide development of COVID-19 therapeutics targeting this complex.

The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.

The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions.

Papain-like protease (PLpro) from SARS-CoV-2 plays essential roles in the replication cycle of the virus. In particular, it preferentially interacts with and cleaves human interferon-stimulated gene 15 (hISG15) to suppress the innate immune response of the host. We used small-angle X-ray and neutron scattering combined with computational techniques to study the mechanism of interaction of SARS-CoV-2 PLpro with hISG15. We showed that hISG15 undergoes a transition from an extended to a compact state after binding to PLpro, a conformation that has not been previously observed in complexes of SARS-CoV-2 PLpro with ISG15 from other species. Furthermore, computational analysis showed significant conformational flexibility in the ISG15 N-terminal domain, suggesting that it is weakly bound to PLpro and supports a binding mechanism that is dominated by the C-terminal ISG15 domain. This study fundamentally improves our understanding of the SARS-CoV-2 deISGylation complex that will help guide development of COVID-19 therapeutics targeting this complex.

Protein dynamics are essential to function. One example of this is the various gating mechanisms within ion channels, which are transmembrane proteins that act as gateways into the cell. Typical ion channels switch between an open and closed state via a conformational transition which is often triggered by an external stimulus, such as ligand binding or pH and voltage differences. The atomic resolution structure of a potassium-selective ion channel named NaK2K has allowed us to observe that a hydro-phobic residue at the bottom of the selectivity filter, Phe92, appears in dual conformations. One of the two conformations of Phe92 restricts the diameter of the exit pore around the selectivity filter, limiting ion flow through the channel, while the other conformation of Phe92 provides a larger-diameter exit pore from the selectivity filter. Thus, it can be concluded that Phe92 acts as a hydro-phobic gate, regulating the flow of ions through the selectivity filter.

Protein dynamics are essential to function. One example of this is the various gating mechanisms within ion channels, which are transmembrane proteins that act as gateways into the cell. Typical ion channels switch between an open and closed state via a conformational transition which is often triggered by an external stimulus, such as ligand binding or pH and voltage differences. The atomic resolution structure of a potassium-selective ion channel named NaK2K has allowed us to observe that a hydro-phobic residue at the bottom of the selectivity filter, Phe92, appears in dual conformations. One of the two conformations of Phe92 restricts the diameter of the exit pore around the selectivity filter, limiting ion flow through the channel, while the other conformation of Phe92 provides a larger-diameter exit pore from the selectivity filter. Thus, it can be concluded that Phe92 acts as a hydro-phobic gate, regulating the flow of ions through the selectivity filter.

Potassium ion channels utilize a highly selective filter to rapidly transport K+ ions across cellular membranes. This selectivity filter is composed of four binding sites which display almost equal electron density in crystal structures with high potassium ion concentrations. This electron density can be interpreted to reflect a superposition of alternating potassium ion and water occupied states or as adjacent potassium ions. Here, we use single wavelength anomalous dispersion (SAD) X-ray diffraction data collected near the potassium absorption edge to show experimentally that all ion binding sites within the selectivity filter are fully occupied by K+ ions. These data support the hypothesis that potassium ion transport occurs by direct Coulomb knock-on, and provide an example of solving the phase problem by K-SAD.

Potassium ion channels utilize a highly selective filter to rapidly transport K+ ions across cellular membranes. This selectivity filter is composed of four binding sites which display almost equal electron density in crystal structures with high potassium ion concentrations. This electron density can be interpreted to reflect a superposition of alternating potassium ion and water occupied states or as adjacent potassium ions. Here, we use single wavelength anomalous dispersion (SAD) X-ray diffraction data collected near the potassium absorption edge to show experimentally that all ion binding sites within the selectivity filter are fully occupied by K+ ions. These data support the hypothesis that potassium ion transport occurs by direct Coulomb knock-on, and provide an example of solving the phase problem by K-SAD.