Studying the Effect of Glycosylation towards SARS-CoV-2 Spike RBD and ACE2 Interaction by Native Mass Spectrometry

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has serious consequences on global public health and social development. The binding of receptor binding domain (RBD) of spike protein to angiotensin converting enzyme 2 (ACE2) on the surface of SARS-CoV-2 host cell initiates the infection progress. Spike and ACE2 are both glycoproteins, the impact of glycosylation on protein structures and protein-protein interactions remains largely elusive. Characterizing the structural and dynamics of protein-protein binding progress will improve mechanism understanding of viral infection and facilitate targeted drug design. Structural mass spectrometry (MS) method is widely used in protein structural studies, providing complementary information to conventional biophysical methods, such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM). Native mass spectrometry (native MS) is an emerging technology that enables the study of intact protein, non-covalent protein-protein, and protein-ligand complexes in their biological state, which can provide structural stability, binding stoichiometry, and spatial arrangement information. Here, native MS was used to examine the interaction between RBD and ACE2 as well as the impact of deglycosylation on the interaction stability of the RBD-ACE2 complex. The results revealed that both RBD and ACE2 are highly glycosylated, ACE2 presents as a dimer while RBD as a monomer, and they form a (RBD-ACE2)2 complex. The conditions of using PNGasc F to remove the N-glycan were optimized. At least two Oglycans including NcuAc(2) and GalNAcC 1) Gal( 1) NcuAc(2) or GlcNAcd ) Gal(l) NeuAc(2) were observed for the N-glycan removed RBD. Furthermore, the stability of the complexes formed by glycosylated and deglycosylated RBD with ACE2 was compared, and the results showed that the removal of N-glycan significantly drops the interaction stability of the RBD-ACE2 complex. Therefore, we recommend that glycosyla-tion should not be removed for structural and functional studies. Additional glycosyla-tion, structural and dynamics studies on Spike (including separated RBD) and ACE2 complexes would help us to understand the process of viral infection, advance drug design and vaccine developments. Nowadays, a comprehensive MS-based toolbox has been developed for the analysis of protein structure, function, and dynamics, including hydrogen-deuterium exchange MS (HDX-MS), native top-down (nTD) MS, cross-linking MS (XL-MS), and covalent labelling MS (CL-MS), etc. Through integrating structural MS methods, more detailed and comprehensive structural information about glycoproteins and their complexes will be uncovered. © 2022 Chinese Society for Mass Spectrometry. All rights reserved.

Studying the Effect of Glycosylation towards SARS-CoV-2 Spike RBD and ACE2 Interaction by Native Mass Spectrometry

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has serious consequences on global public health and social development. The binding of receptor binding domain (RBD) of spike protein to angiotensin converting enzyme 2 (ACE2) on the surface of SARS-CoV-2 host cell initiates the infection progress. Spike and ACE2 are both glycoproteins, the impact of glycosylation on protein structures and protein-protein interactions remains largely elusive. Characterizing the structural and dynamics of protein-protein binding progress will improve mechanism understanding of viral infection and facilitate targeted drug design. Structural mass spectrometry (MS) method is widely used in protein structural studies, providing complementary information to conventional biophysical methods, such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM). Native mass spectrometry (native MS) is an emerging technology that enables the study of intact protein, non-covalent protein-protein, and protein-ligand complexes in their biological state, which can provide structural stability, binding stoichiometry, and spatial arrangement information. Here, native MS was used to examine the interaction between RBD and ACE2 as well as the impact of deglycosylation on the interaction stability of the RBD-ACE2 complex. The results revealed that both RBD and ACE2 are highly glycosylated, ACE2 presents as a dimer while RBD as a monomer, and they form a (RBD-ACE2)2 complex. The conditions of using PNGasc F to remove the N-glycan were optimized. At least two Oglycans including NcuAc(2) and GalNAcC 1) Gal( 1) NcuAc(2) or GlcNAcd ) Gal(l) NeuAc(2) were observed for the N-glycan removed RBD. Furthermore, the stability of the complexes formed by glycosylated and deglycosylated RBD with ACE2 was compared, and the results showed that the removal of N-glycan significantly drops the interaction stability of the RBD-ACE2 complex. Therefore, we recommend that glycosyla-tion should not be removed for structural and functional studies. Additional glycosyla-tion, structural and dynamics studies on Spike (including separated RBD) and ACE2 complexes would help us to understand the process of viral infection, advance drug design and vaccine developments. Nowadays, a comprehensive MS-based toolbox has been developed for the analysis of protein structure, function, and dynamics, including hydrogen-deuterium exchange MS (HDX-MS), native top-down (nTD) MS, cross-linking MS (XL-MS), and covalent labelling MS (CL-MS), etc. Through integrating structural MS methods, more detailed and comprehensive structural information about glycoproteins and their complexes will be uncovered. © 2022 Chinese Society for Mass Spectrometry. All rights reserved.

Allosteric Inhibition of the SARS-CoV-2 Main Protease: Insights from Mass Spectrometry Based Assays

The SARS-CoV-2 main protease (Mpm) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease. Here, we used native mass spectrometry to characterize the functional unit of Mpro. Analysis of the monomer/dimer equilibria reveals a dissociation constant of Kd = 0.14± 0.03 pM, indicating MPro has a strong preference to dimerize in solution. We characterized substrate turnover rates by following temporal changes in the enzyme-substrate com- plexes, and screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the dimer, slow the rate of substrate processing by ~35 %. This informa- tion, together with analysis of the x-ray crystal structures, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity. . © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Backbone chemical shift assignments for the SARS-CoV-2 non-structural protein Nsp9: intermediate (ms - µs) dynamics in the C-terminal helix at the dimer interface.

The Betacoronavirus SARS-CoV-2 non-structural protein Nsp9 is a 113-residue protein that is essential for viral replication, and consequently, a potential target for the development of therapeutics against COVID19 infections. To capture insights into the dynamics of the protein's backbone in solution and accelerate the identification and mapping of ligand-binding surfaces through chemical shift perturbation studies, the backbone 1H, 13C, and 15N NMR chemical shifts for Nsp9 have been extensively assigned. These assignments were assisted by the preparation of an ~ 70% deuterated sample and residue-specific, 15N-labelled samples (V, L, M, F, and K). A major feature of the assignments was the "missing" amide resonances for N96-L106 in the 1H-15N HSQC spectrum, a region that comprises almost the complete C-terminal α-helix that forms a major part of the homodimer interface in the crystal structure of SARS-CoV-2 Nsp9, suggesting this region either undergoes intermediate motion in the ms to µs timescale and/or is heterogenous. These "missing" amide resonances do not unambiguously appear in the 1H-15N HSQC spectrum of SARS-CoV-2 Nsp9 collected at a concentration of 0.0007 mM. At this concentration, at the detection limit, native mass spectrometry indicates the protein is exclusively in the monomeric state, suggesting the intermediate motion in the C-terminal of Nsp9 may be due to intramolecular dynamics. Perhaps this intermediate ms to µs timescale dynamics is the physical basis for a previously suggested "fluidity" of the C-terminal helix that may be responsible for homophilic (Nsp9-Nsp9) and postulated heterophilic (Nsp9-Unknown) protein-protein interactions.

Native Mass Spectrometry-Based Screening for Optimal Sample Preparation in Single-Particle Cryo-EM.

Recent advances in single-particle cryogenic electron microscopy (cryo-EM) have enabled the structural determination of numerous protein assemblies at high resolution, yielding unprecedented insights into their function. However, despite its extraordinary capabilities, cryo-EM remains time-consuming and resource-intensive. It is therefore beneficial to have a means for rapidly assessing and optimizing the quality of samples prior to lengthy cryo-EM analyses. To do this, we have developed a native mass spectrometry (nMS) platform that provides rapid feedback on sample quality and highly streamlined biochemical screening. Because nMS enables accurate mass analysis of protein complexes, it is well suited to routine evaluation of the composition, integrity, and homogeneity of samples prior to their plunge-freezing on EM grids. We demonstrate the utility of our nMS-based platform for facilitating cryo-EM studies using structural characterizations of exemplar bacterial transcription complexes as well as the replication-transcription assembly from the SARS-CoV-2 virus that is responsible for the COVID-19 pandemic.

Processing of the SARS-CoV pp1a/ab nsp7-10 region.

Severe acute respiratory syndrome coronavirus is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex, essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease Mpro and subsequent complex formation of the released nsp's. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2 : 2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.

Allosteric Inhibition of the SARS-CoV-2 Main Protease: Insights from Mass Spectrometry Based Assays*.

The SARS-CoV-2 main protease (Mpro ) cleaves along the two viral polypeptides to release non-structural proteins required for viral replication. MPro is an attractive target for antiviral therapies to combat the coronavirus-2019 disease. Here, we used native mass spectrometry to characterize the functional unit of Mpro . Analysis of the monomer/dimer equilibria reveals a dissociation constant of Kd =0.14±0.03 µM, indicating MPro has a strong preference to dimerize in solution. We characterized substrate turnover rates by following temporal changes in the enzyme-substrate complexes, and screened small molecules, that bind distant from the active site, for their ability to modulate activity. These compounds, including one proposed to disrupt the dimer, slow the rate of substrate processing by ≈35 %. This information, together with analysis of the x-ray crystal structures, provides a starting point for the development of more potent molecules that allosterically regulate MPro activity.