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.
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.
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揭示了N-聚糖占用率在峰值和血管紧张素合作组装中的作用，转化酶2络合物：来自糖制造和天然质谱的见解。
DOI：
10.1021/jacs.3c00291
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发表时间：
2023-04-12
影响因子：
15
通讯作者：
Robinson, Carol V.
中科院分区：
文献类型：
--
作者：
El-Baba, Tarick J.;Lutomski, Corinne A.;Burnap, Sean A.;Bolla, Jani R.;Baker, Lindsay A.;Baldwin, Andrew J.;Struwe, Weston B.;Robinson, Carol V.
SARS-CoV-2刺突蛋白和ACE 2之间的相互作用是我们这个时代最受关注的反应之一。然而，关于聚糖对介导ACE 2二聚化和下游与Spike相互作用的影响仍然存在问题。在这里，我们通过将糖工程策略与高分辨率天然质谱（MS）相结合来解决这些未回答的问题，以研究N-聚糖占据对多个Spike-ACE 2复合物组装的影响。我们证实了完整的刺突三聚体具有全部66个N-连接位点被占据。对于单体ACE 2，所有七个N-连接聚糖位点都被不同程度地占据;六个位点具有>90%的占据率，而第七个位点（Asn 690）仅被部分占据（约30%）。通过解析ACE 2上的糖型，我们破译了每个N-聚糖对ACE 2二聚化的影响。出乎意料的是，我们发现Asn 432在介导二聚化中起作用，这一结果得到了定点突变的证实。我们还发现糖基化的二聚ACE 2和Spike三聚体形成具有多个化学计量的复合物（Spike-ACE 2和Spike 2-ACE 2），其解离常数（Kds）分别为1000和<100 nM。比较这些值表明，正协同性可驱动ACE 2二聚体与多个刺突三聚体复合。总体而言，我们的研究结果表明，占有率在介导ACE 2二聚体和刺突三聚体之间的相互作用中具有关键的调节作用。更一般地说，由于可溶性ACE 2（sACE 2）保留了完整的SARS-CoV-2相互作用位点，因此ACE 2二聚化中糖基化的重要性以及Spike和ACE 2组装成高级寡聚体的倾向是开发中和病毒策略的重要分子细节。
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 (Kds) 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.
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影响因子：
4.9
作者：
Burnap, Sean A.;Struwe, Weston B.
通讯作者：
Struwe, Weston B.
影响因子：
64.8
作者：
Li W;Moore MJ;Vasilieva N;Sui J;Wong SK;Berne MA;Somasundaran M;Sullivan JL;Luzuriaga K;Greenough TC;Choe H;Farzan M
通讯作者：
Farzan M
影响因子：
12.4
作者：
通讯作者：
--
影响因子：
14.8
作者：
Chaudhary, Sarika;Pak, John E.;Stroud, Robert M.
通讯作者：
Stroud, Robert M.
DOI：
10.1126/science.abc0870
发表时间：
2020-09-04
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Chan KK;Dorosky D;Sharma P;Abbasi SA;Dye JM;Kranz DM;Herbert AS;Procko E
通讯作者：
Procko E