Uncovering the role of N-glycan occupancy on the cooperative assembly of spike and angiotensin converting enzyme 2 complexes: insights from glycoengineering and native mass spectrometry

El-Baba, TJ; Lutomski, CA; Burnap, SA; Bolla, JR; Baker, LA; Baldwin, AJ; Struwe, WB; Robinson, CV

Journal article

Uncovering the role of N-glycan occupancy on the cooperative assembly of spike and angiotensin converting enzyme 2 complexes: insights from glycoengineering and native mass spectrometry

Abstract:: Interactions between the SARS-CoV-2 Spike protein and ACE2 are one of the most scrutinized reactions of our time. Yet, questions remain as to the impact of glycans on mediating ACE2 dimerization and downstream interactions with Spike. Here, we address these unanswered questions by combining a glycoengineering strategy with high-resolution native mass spectrometry (MS) to investigate the impact of N-glycan occupancy on the assembly of multiple Spike-ACE2 complexes. We confirmed that intact Spike trimers have all 66 N-linked sites occupied. For monomeric ACE2, all seven N-linked glycan sites are occupied to various degrees; six sites have >90% occupancy, while the seventh site (Asn690) is only partially occupied (∼30%). By resolving the glycoforms on ACE2, we deciphered the influence of each N-glycan on ACE2 dimerization. Unexpectedly, we found that Asn432 plays a role in mediating dimerization, a result confirmed by site-directed mutagenesis. We also found that glycosylated dimeric ACE2 and Spike trimers form complexes with multiple stoichiometries (Spike-ACE2 and Spike₂-ACE2) with dissociation constants (K_ds) of ∼500 and <100 nM, respectively. Comparing these values indicates that positive cooperativity may drive ACE2 dimers to complex with multiple Spike trimers. Overall, our results show that occupancy has a key regulatory role in mediating interactions between ACE2 dimers and Spike trimers. More generally, since soluble ACE2 (sACE2) retains an intact SARS-CoV-2 interaction site, the importance of glycosylation in ACE2 dimerization and the propensity for Spike and ACE2 to assemble into higher oligomers are molecular details important for developing strategies for neutralizing the virus.

Publication status:: Published

Peer review status:: Peer reviewed

Actions

Email

Email this record

Send the bibliographic details of this record to your email address.

Your Email
Please enter the email address that the record information will be sent to.

-
Your message (optional)
Please add any additional information to be included within the email.
Cite

Cite this record

APA Style

El-Baba, T. J., Lutomski, C. A., Burnap, S. A., Bolla, J. R., Baker, L. A., Baldwin, A. J., Struwe, W. B., & Robinson, C. V. (2023). Uncovering the role of N-glycan occupancy on the cooperative assembly of spike and angiotensin converting enzyme 2 complexes: insights from glycoengineering and native mass spectrometry. Journal of the American Chemical Society, 145(14), 8021–8032.

MLA Style

El-Baba, T. J., et al. “Uncovering the Role of N-Glycan Occupancy on the Cooperative Assembly of Spike and Angiotensin Converting Enzyme 2 Complexes: Insights from Glycoengineering and Native Mass Spectrometry.” Journal of the American Chemical Society, vol. 145, no. 14, American Chemical Society, 2023, pp. 8021–32.

Chicago Style

El-Baba, TJ, CA Lutomski, SA Burnap, JR Bolla, LA Baker, AJ Baldwin, WB Struwe, and CV Robinson. 2023. “Uncovering the Role of N-Glycan Occupancy on the Cooperative Assembly of Spike and Angiotensin Converting Enzyme 2 Complexes: Insights from Glycoengineering and Native Mass Spectrometry.” Journal of the American Chemical Society 145 (14): 8021–32.
Share
Print