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, noncovalent 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 PNGase F to remove the N-glycan were optimized. At least two O-glycans including NeuAc(2) and GalNAc(1)Gal(1)NeuAc(2) or GlcNAc(1)Gal(1)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 glycosylation should not be removed for structural and functional studies. Additional glycosylation, 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.