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Highlights of Coronavirus Structural Studies

29 Mar 2021

Heterogeneity of Glycan Processing on Trimeric SARS-CoV-2 Spike Protein Revealed by Charge Detection Mass Spectrometry (J. Amer. Chem. Soc.)

The heterogeneity associated with glycosylation of the 66 N-glycan sites on the protein trimer making up the spike (S) region of the SARS-CoV-2 virus has been assessed by charge detection mass spectrometry (CDMS). CDMS allows simultaneous measurement of the mass-to-charge ratio and charge of individual ions, so that mass distributions can be determined for highly heterogeneous proteins such as the heavily glycosylated S protein trimer. The CDMS results are compared to recent glycoproteomics studies of the structure and abundance of glycans at specific sites. Interestingly, average glycan masses determined by “top-down” CDMS measurements are 35–47% larger than those obtained from the “bottom-up” glycoproteomics studies, suggesting that the glycoproteomic measurements underestimated the abundances of larger, more-complex glycans. Moreover, the distribution of glycan masses determined by CDMS is much broader than the distribution expected from the glycoproteomics studies, assuming that glycan processing on each trimer is not correlated. The breadth of the glycan mass distribution therefore indicates heterogeneity in the extent of glycan processing of the S protein trimers, with some trimers being much more heavily processed than others. This heterogeneity may have evolved as a way of further confounding the host’s immune system.

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Reader's Corner Archive

4 Aug 2021

Cryo-EM structure and dynamics of the green-light absorbing proteorhodopsin (Nature Communications)

The green-light absorbing proteorhodopsin (GPR) is the archetype of bacterial light-driven proton pumps. Here, we present the 2.9 Å cryo-EM structure of pentameric GPR, resolving important residues of the proton translocation pathway and the oligomerization interface. Superposition with the structure of a close GPR homolog and molecular dynamics simulations reveal conformational variations, which regulate the solvent access to the intra- and extracellular half channels harbouring the primary proton donor E109 and the proposed proton release group E143. We provide a mechanism for the structural rearrangements allowing hydration of the intracellular half channel, which are triggered by changing the protonation state of E109. Functional characterization of selected mutants demonstrates the importance of the molecular organization around E109 and E143 for GPR activity. Furthermore, we present evidence that helices involved in the stabilization of the protomer interfaces serve as scaffolds for facilitating the motion of the other helices. Combined with the more constrained dynamics of the pentamer compared to the monomer, these observations illustrate the previously demonstrated functional significance of GPR oligomerization. Overall, this work provides molecular insights into the structure, dynamics and function of the proteorhodopsin family that will benefit the large scientific community employing GPR as a model protein.

4 Aug 2021

INI1/SMARCB1 Rpt1 domain mimics TAR RNA in binding to integrase to facilitate HIV-1 replication (Nature Communications)

INI1/SMARCB1 binds to HIV-1 integrase (IN) through its Rpt1 domain and exhibits multifaceted role in HIV-1 replication. Determining the NMR structure of INI1-Rpt1 and modeling its interaction with the IN-C-terminal domain (IN-CTD) reveal that INI1-Rpt1/IN-CTD interface residues overlap with those required for IN/RNA interaction. Mutational analyses validate our model and indicate that the same IN residues are involved in both INI1 and RNA binding. INI1-Rpt1 and TAR RNA compete with each other for IN binding with similar IC50 values. INI1-interaction-defective IN mutant viruses are impaired for incorporation of INI1 into virions and for particle morphogenesis. Computational modeling of IN-CTD/TAR complex indicates that the TAR interface phosphates overlap with negatively charged surface residues of INI1-Rpt1 in three-dimensional space, suggesting that INI1-Rpt1 domain structurally mimics TAR. This possible mimicry between INI1-Rpt1 and TAR explains the mechanism by which INI1/SMARCB1 influences HIV-1 late events and suggests additional strategies to inhibit HIV-1 replication. HIV-1 integrase (IN) binds the host factor INI1/SMARCB1, which is required at multiple stages of HIV-1 replication. Here, the authors show that the same IN residues are involved in INI1 and RNA binding and in influencing particle morphogenesis and suggest that the IN-binding INI1 domain is structurally similar to HIV TAR RNA.

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