10 Nov 2021

Molecular basis of immune evasion by the Delta and Kappa SARS-CoV-2 variants (Science)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission leads to the emergence of variants, including the B.1.617.2 (Delta) variant of concern which is causing a new wave of infections and has become globally dominant. D. Veesler et.al. show that these variants dampen the in vitro potency of vaccine-elicited serum neutralizing antibodies and provide a structural framework for describing their immune evasion. Mutations in the B.1.617.1 (Kappa) and B.1.617.2 (Delta) spike glycoproteins abrogate recognition by several monoclonal antibodies via alteration of key antigenic sites, including remodeling of the B.1.617.2 (Delta) N-terminal domain. They determined electron cryo-microscopy (cryo-EM) structures of the B.1.617.1 and B.1.617.2 S glycoproteins to gain insights into their reduced antibody sensitivity and provide a structural framework to understand the role of shared and distinct mutations.  The ACE2 binding affinities of the B.1.617.1 (Kappa) and B.1.617.2 (Delta) receptor-binding domains are comparable to the Wuhan-Hu-1 isolate whereas B.1.617.2+ (Delta+) exhibits markedly reduced affinity.

8 Nov 2021

Crystal structure of SARS-CoV-2 main protease in complex with protease inhibitor PF-07321332 (Protein & Cell)

The crystal structure of SARS-CoV-2 M^pro in complex with PF-07321332 at 1.6 Å resolution was determined. PF-07321332 is a reversible covalent inhibitor carrying a nitrile warhead that targets SARS-CoV-2 M^pro. The overall structure of SARS-CoV-2 M^pro is a functional dimer, consistent with previous findings. The 1.6-Å crystal structure of SARS-CoV-2 M^pro in complex with the protease inhibitor PF-07321332 reveals critical insight for pharmaceutical development. With the emergence of SARS-CoV-2 variants with immune evasion, therapeutics targeting a conserved but essential viral target among the variants may synergize the COVID-19 vaccines. The improved oral bioavailability would enhance the clinical utility of PF-07321332 as a therapeutic to prevent progression among early-stage non-hospitalized patients or as a prophylaxis in the pre- and post-exposure setting. Further optimization of such covalent peptidomimetic inhibitors would generate potent drugs to contain the current COVID-19 pandemic and increase public health preparedness for potential future pandemic.

8 Nov 2021

NMR Spectroscopy of the Main Protease of SARS-CoV-2 and Fragment-Based Screening Identify Three Protein Hotspots and an Antiviral Fragment (Angewandte Chemie Int. Ed)

The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, J. Charton, X. Hanoulle et. al. studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. They performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp and constitutes a strong basis to assist in developing potent 3CLp inhibitors.

24 Jan 2023

Structural visualization of the tubulin folding pathway directed by human chaperonin TRiC/CCT (Cell)

The ATP-dependent ring-shaped chaperonin TRiC/CCT is essential for cellular proteostasis. To uncover why some eukaryotic proteins can only fold with TRiC assistance, we reconstituted the folding of β-tubulin using human prefoldin and TRiC. We find unstructured β-tubulin is delivered by prefoldin to the open TRiC chamber followed by ATP-dependent chamber closure. Cryo-EM resolves four near-atomic-resolution structures containing progressively folded β-tubulin intermediates within the closed TRiC chamber, culminating in native tubulin. This substrate folding pathway appears closely guided by site-specific interactions with conserved regions in the TRiC chamber. Initial electrostatic interactions between the TRiC interior wall and both the folded tubulin N domain and its C-terminal E-hook tail establish the native substrate topology, thus enabling C-domain folding. Intrinsically disordered CCT C termini within the chamber promote subsequent folding of tubulin’s core and middle domains and GTP-binding. Thus, TRiC’s chamber provides chemical and topological directives that shape the folding landscape of its obligate substrates.

24 Jan 2023

A single 2′-O-methylation of ribosomal RNA gates assembly of a functional ribosome (Nat. Mol. Struct. Biol.)

RNA modifications are widespread in biology and abundant in ribosomal RNA. However, the importance of these modifications is not well understood. We show that methylation of a single nucleotide, in the catalytic center of the large subunit, gates ribosome assembly. Massively parallel mutational scanning of the essential nuclear GTPase Nog2 identified important interactions with rRNA, particularly with the 2′-O-methylated A-site base Gm2922. We found that methylation of G2922 is needed for assembly and efficient nuclear export of the large subunit. Critically, we identified single amino acid changes in Nog2 that completely bypass dependence on G2922 methylation and used cryoelectron microscopy to directly visualize how methylation flips Gm2922 into the active site channel of Nog2. This work demonstrates that a single RNA modification is a critical checkpoint in ribosome biogenesis, suggesting that such modifications can play an important role in regulation and assembly of macromolecular machines.

24 Jan 2023

Correlation between the binding affinity and the conformational entropy of nanobody SARS-CoV-2 spike protein complexes (PNAS)

Camelid single-domain antibodies, also known as nanobodies, can be readily isolated from naïve libraries for specific targets but often bind too weakly to their targets to be immediately useful. Laboratory-based genetic engineering methods to enhance their affinity, termed maturation, can deliver useful reagents for different areas of biology and potentially medicine. Using the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and a naïve library, we generated closely related nanobodies with micromolar to nanomolar binding affinities. By analyzing the structure–activity relationship using X-ray crystallography, cryoelectron microscopy, and biophysical methods, we observed that higher conformational entropy losses in the formation of the spike protein–nanobody complex are associated with tighter binding.To investigate this, we generated structural ensembles of the different complexes from electron microscopy maps and correlated the conformational fluctuations with binding affinity. This insight guided the engineering of a nanobody with improved affinity for the spike protein.

24 Jan 2023

Structural basis for Parkinson's disease linked LRRK2's binding to microtubules (Nat. Mo. Struct. Biol.)

Leucine-rich repeat kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinson’s disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an association enhanced by PD mutations. We report a cryo-EM structure of the catalytic half of LRRK2, containing its kinase, in a closed conformation, and GTPase domains, bound to microtubules. We also report a structure of the catalytic half of LRRK1, which is closely related to LRRK2 but is not linked to PD. Although LRRK1’s structure is similar to that of LRRK2, we find that LRRK1 does not interact with microtubules. Guided by these structures, we identify amino acids in LRRK2’s GTPase that mediate microtubule binding; mutating them disrupts microtubule binding in vitro and in cells, without affecting LRRK2’s kinase activity. Our results have implications for the design of therapeutic LRRK2 kinase inhibitors.

24 Jan 2023

Structural basis for the severe adverse interaction of sofosbuvir and amiodarone on L-type Cav channels (Cell)

Drug-drug interaction of the antiviral sofosbuvir and the antiarrhythmics amiodarone has been reported to cause fatal heartbeat slowing. Sofosbuvir and its analog, MNI-1, were reported to potentiate the inhibition of cardiomyocyte calcium handling by amiodarone, which functions as a multi-channel antagonist, and implicate its inhibitory effect on L-type Ca_v channels, but the molecular mechanism has remained unclear. Here we present systematic cryo-EM structural analysis of Ca_v1.1 and Ca_v1.3 treated with amiodarone or sofosbuvir alone, or sofosbuvir/MNI-1 combined with amiodarone. Whereas amiodarone alone occupies the dihydropyridine binding site, sofosbuvir is not found in the channel when applied on its own. In the presence of amiodarone, sofosbuvir/MNI-1 is anchored in the central cavity of the pore domain through specific interaction with amiodarone and directly obstructs the ion permeation path. Our study reveals the molecular basis for the physical, pharmacodynamic interaction of two drugs on the scaffold of Ca_vchannels.

9 Dec 2022

Cryo-electron tomography: A long journey to the inner space of cells (Cell)

Cryogenic electron tomography (cryo-ET) is the application of tomographic principles of data acquisition and reconstruction to frozen-hydrated biological specimens. It combines a close-to-life preservation of cellular structures with the power of high-resolution three-dimensional imaging, which allows us to study the molecular architecture of cells, or their molecular sociology, in unprecedented detail. The journey of cryo-ET to the inner space of cells has been a long and tedious one. In the paper, some of the limitations of the method will be discussed along with prospects to overcome them.

9 Dec 2022

Uncovering structural ensembles from single-particle cryo-EM data using cryoDRGN (Nature Protocols)

Single-particle cryogenic electron microscopy (cryo-EM) has emerged as a powerful technique to visualize the structural landscape sampled by a protein complex. However, algorithmic and computational bottlenecks in analyzing heterogeneous cryo-EM datasets have prevented the full realization of this potential. CryoDRGN is a machine learning system for heterogeneous cryo-EM reconstruction of proteins and protein complexes from single-particle cryo-EM data. Central to this approach is a deep generative model for heterogeneous cryo-EM density maps, which we empirically find is effective in modeling both discrete and continuous forms of structural variability. Once trained, cryoDRGN is capable of generating an arbitrary number of 3D density maps, and thus interpreting the resulting ensemble is a challenge. Here, we showcase interactive and automated processing approaches for analyzing cryoDRGN results. Specifically, we detail a step-by-step protocol for the analysis of an existing assembling 50S ribosome dataset, including preparation of inputs, network training and visualization of the resulting ensemble of density maps. Additionally, we describe and implement methods to comprehensively analyze and interpret the distribution of volumes with the assistance of an associated atomic model. This protocol is appropriate for structural biologists familiar with processing single-particle cryo-EM datasets and with moderate experience navigating Python and Jupyter notebooks. It requires 3–4 days to complete. CryoDRGN is open source software that is freely available.

7 Dec 2022

Commercial gigahertz-class NMR magnets (Superconductor Science and Technology)

Nuclear magnetic resonance (NMR) spectroscopy is a wide-spread analytical technique which is used in a large range of different fields, such as quality control, food analysis, material science and structural biology. In the widest sense, NMR is an analytical technique to determine the structure of molecules. At the time of writing this manuscript, commercial NMR spectrometers with a proton resonance frequency ⩾900 MHz are only available from Bruker. In 2019, Bruker installed the first 1.1 GHz (25.8 T) NMR spectrometer at the St. Jude Children Research Hospital in Memphis, Tennessee, followed by the installation of the first 1.2 GHz (28.2 T) NMR spectrometer at the University of Florence in Italy in 2020. These were the first commercial NMR spectrometers operating at magnetic fields in excess of what can be achieved with conventional low temperature superconductors, and which depend on high temperature superconductors to generate the required magnetic field. In this paper, the requirements on commercial NMR magnets are discussed and the history of high-field NMR magnets is reviewed. Bruker's R&D program for 1.1 and 1.2 GHz NMR magnets and spectrometers will be described, and some of the key properties of these first commercial NMR magnets with high-temperature superconductors are reported.