13 Apr 2021

Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape (Science)

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread, with devastating consequences. For passive immunization efforts, nanobodies have size and cost advantages over conventional antibodies. In this study, Wu, N. et al. generated four neutralizing nanobodies that target the receptor binding domain of the SARS-CoV-2 spike protein. They used x-ray crystallography and cryo-electron microscopy to define two distinct binding epitopes. On the basis of these structures, they engineered multivalent nanobodies with more than 100 times the neutralizing activity of monovalent nanobodies. Biparatopic nanobody fusions suppressed the emergence of escape mutants. Several nanobody constructs neutralized through receptor binding competition, whereas other monovalent and biparatopic nanobodies triggered aberrant activation of the spike fusion machinery. These premature conformational changes in the spike protein forestalled productive fusion and rendered the virions noninfectious.

13 Apr 2021

Bat and pangolin coronavirus spike glycoprotein structures provide insights into SARS-CoV-2 evolution (Nature Communications)

In recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (S) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. Here we determined the cryo-EM structures of the spikes from bat (RaTG13) and pangolin (PCoV_GX) coronaviruses, which are closely related to SARS-CoV-2. All three receptor-binding domains (RBDs) of these two spike trimers are in the "down" conformation, indicating they are more prone to adopt the receptor-binding inactive state. However, Wang, XQ et al. found that the PCoV_GX, but not the RaTG13, spike is comparable to the SARS-CoV-2 spike in binding the human ACE2 receptor and supporting pseudovirus cell entry. They further identified critical residues in the RBD underlying different activities of the RaTG13 and PCoV_GX/SARS-CoV-2 spikes. These results collectively indicate that tight RBD-ACE2 binding and efficient RBD conformational sampling are required for the evolution of SARS-CoV-2 to gain highly efficient infection.

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.

16 Aug 2021

Cryo-EM structure of Helicobacter pylori urease with an inhibitor in the active site at 2.0 Å resolution (Nature Communications)

Infection of the human stomach by Helicobacter pylori remains a worldwide problem and greatly contributes to peptic ulcer disease and gastric cancer. Without active intervention approximately 50% of the world population will continue to be infected with this gastric pathogen. Current eradication, called triple therapy, entails a proton-pump inhibitor and two broadband antibiotics, however resistance to either clarithromycin or metronidazole is greater than 25% and rising. Therefore, there is an urgent need for a targeted, high-specificity eradication drug. Gastric infection by H. pylori depends on the expression of a nickel-dependent urease in the cytoplasm of the bacteria. Here, H. Luecke et. al. report the 2.0 Å resolution structure of the 1.1 MDa urease in complex with an inhibitor by cryo-electron microscopy and compare it to a β-mercaptoethanol-inhibited structure at 2.5 Å resolution. The structural information is of sufficient detail to aid in the development of inhibitors with high specificity and affinity.

16 Aug 2021

Cryo-EM structure of the human MT1–Gi signaling complex (Nature Structural and Molecular Biology)

Melatonin receptors (MT1 and MT2) transduce inhibitory signaling by melatonin (N-acetyl-5-methoxytryptamine), which is associated with sleep induction and circadian rhythm modulation. Although recently reported crystal structures of ligand-bound MT1 and MT2elucidated the basis of ligand entry and recognition, the ligand-induced MT1 rearrangement leading to Gi-coupling remains unclear. Here T. Nishizawa, O. Nurek et. al. report a cryo-EM structure of the human MT1–Gi signaling complex at 3.3 Å resolution, revealing melatonin-induced conformational changes propagated to the G-protein-coupling interface during activation. In contrast to other Gi-coupled receptors, MT1 exhibits a large outward movement of TM6, which is considered a specific feature of Gs-coupled receptors. Structural comparison of Gi and Gscomplexes demonstrated conformational diversity of the C-terminal entry of the Gi protein, suggesting loose and variable interactions at the end of the α5 helix of Gi protein. These notions, together with their biochemical and computational analyses, highlight variable binding modes of Gαi and provide the basis for the selectivity of G-protein signaling.

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.

3 Aug 2021

Will electron diffraction revolutionize drug development (Cambridge Crystallographic Data Center blog)

Microcrystal electron diffraction (MicroED or 3D ED) uses a beam of electrons rather than X-rays to obtain structures. In a recent paper, a team of researchers led by NanoImaging Services presented a new advancement in drug development using electron diffraction for when X-ray diffraction (XRD) may not be an option. In this blog, we talk with author Dr Jessica Bruhn, Scientific Group Leader - MicroED at NanoImaging Services about the team’s findings.

3 Aug 2021

Small Molecule Microcrystal Electron Diffraction for the Pharmaceutical Industry–Lessons Learned From Examining Over Fifty Samples (Frontiers in Molecular Biosciences)

The emerging field of microcrystal electron diffraction (MicroED) is of great interest to industrial researchers working in the drug discovery and drug development space. The promise of being able to routinely solve high-resolution crystal structures without the need to grow large crystals is very appealing. Despite MicroED’s exciting potential, adoption across the pharmaceutical industry has been slow, primarily owing to a lack of access to specialized equipment and expertise. Here Bridget Carragher, Jessica Bruhn et. al.  present their experience building a small molecule MicroED service pipeline for members of the pharmaceutical industry. In the past year, they have examined more than fifty small molecule samples submitted by our clients, the majority of which have yielded data suitable for structure solution. They also detail the experience determining small molecule MicroED structures of pharmaceutical interest and offer some insights into the typical experimental outcomes. This experience has led them to conclude that small molecule MicroED adoption will continue to grow within the pharmaceutical industry where it is able to rapidly provide structures inaccessible by other methods.

15 Jul 2021

Analysis of Zika virus capsid-Aedes aegypti mosquito interactome reveals pro-viral host factors critical for establishing infection (Nature Communications)

The escalating global prevalence of arboviral diseases emphasizes the need to improve our understanding of their biology. Research in this area has been hindered by the lack of molecular tools for studying virus-mosquito interactions. Here, authors develop an Aedes aegypti cell line which stably expresses Zika virus (ZIKV) capsid proteins in order to study virus-vector protein-protein interactions through quantitative label-free proteomics. They identify 157 interactors and show that eight have potentially pro-viral activity during ZIKV infection in mosquito cells. Notably, silencing of transitional endoplasmic reticulum protein TER94 prevents ZIKV capsid degradation and significantly reduces viral replication. Similar results are observed if the TER94 ortholog (VCP) functioning is blocked with inhibitors in human cells. In addition, they show that an E3 ubiquitin-protein ligase, UBR5, mediates the interaction between TER94 and ZIKV capsid. The study demonstrates a pro-viral function for TER94/VCP during ZIKV infection that is conserved between human and mosquito cells.

15 Jul 2021

A unified route for flavivirus structures uncovers essential pocket factors conserved across pathogenic viruses (Nature Communications)

The epidemic emergence of relatively rare and geographically isolated flaviviruses adds to the ongoing disease burden of viruses such as dengue. Structural analysis is key to understand and combat these pathogens. Here, the authors present a chimeric platform based on an insect-specific flavivirus for the safe and rapid structural analysis of pathogenic viruses. They use this approach to resolve the architecture of two neurotropic viruses and a structure of dengue virus at 2.5 angstrom, the highest resolution for an enveloped virion. These reconstructions allow improved modelling of the stem region of the envelope protein, revealing two lipid-like ligands within highly conserved pockets. We show that these sites are essential for viral growth and important for viral maturation. These findings define a hallmark of flavivirus virions and a potential target for broad-spectrum antivirals and vaccine design. Authors anticipate the chimeric platform to be widely applicable for investigating flavivirus biology. Understanding virus assembly could identify potential drug targets. The authors use a safe and efficient method to solve pathogenic flavivirus structures, revealing two lipid-like ligands within highly conserved pockets of the stem region of envelope protein that are important for virus maturation.