6 Apr 2022

Deep learning guided optimization of human antibody against SARS-CoV-2 variants with broad neutralization (PNAS)

The ability of viruses to mutate and evade the human immune system and neutralizing antibodies remains an obstacle to antiviral and vaccine development. Many neutralizing antibodies, including some approved for emergency use authorization (EUA), reduced or lost activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Here, we introduce a geometric deep learning algorithm that efficiently enhances antibody affinity to achieve broader and more potent neutralizing activity against such variants. We demonstrate the utility of our approach on a human antibody P36-5D2, which is effective against SARS-CoV-2 Alpha, Beta, and Gamma but not Delta. We show that our geometric neural network model optimizes this antibody's complementaritydetermining region (CDR) sequences to improve its binding affinity against multiple SARS-CoV-2 variants. Through iterative optimization of the CDR regions and experimental measurements, we enable expanded antibody breadth and improved potency by similar to 10to 600-fold against SARS-CoV-2 variants, including Delta. We have also demonstrated that our approach can identify CDR changes that alleviate the impact of two Omicron mutations on the epitope. These results highlight the power of our deep learning approach in antibody optimization and its potential application to engineering other protein molecules. Our optimized antibodies can potentially be developed into antibody drug candidates for current and emerging SARS-CoV-2 variants.

6 Apr 2022

Ensemble cryo-EM reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication–transcription complex (Nature Structural and Molecular Biology)

The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA-dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication–transcription complex or RTC) associated with two copies of nsp13 (nsp13₂–RTC). One copy of nsp13 interacts with the template-RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backward on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13. Here we use cryogenic-electron microscopy and molecular dynamics simulations to analyze the nsp13₂–RTC, revealing four distinct conformational states of the helicases. The results indicate a mechanism for the nsp13₂–RTC to turn backtracking on and off, using an allosteric mechanism to switch between RNA synthesis or backtracking in response to stimuli at the RdRp active site.

6 Apr 2022

Structural and biochemical rationale for enhanced spike protein fitness in delta and kappa SARS-CoV-2 variants (Nature Communications)

The Delta and Kappa variants of SARS-CoV-2 co-emerged in India in late 2020, with the Delta variant underlying the resurgence of COVID-19, even in countries with high vaccination rates. In this study, we assess structural and biochemical aspects of viral fitness for these two variants using cryo-electron microscopy (cryo-EM), ACE2-binding and antibody neutralization analyses. Both variants demonstrate escape of antibodies targeting the N-terminal domain, an important immune hotspot for neutralizing epitopes. Compared to wild-type and Kappa lineages, Delta variant spike proteins show modest increase in ACE2 affinity, likely due to enhanced electrostatic complementarity at the RBD-ACE2 interface, which we characterize by cryo-EM. Unexpectedly, Kappa variant spike trimers form a structural head-to-head dimer-of-trimers assembly, which we demonstrate is a result of the E484Q mutation and with unknown biological implications. The combination of increased antibody escape and enhanced ACE2 binding provides an explanation, in part, for the rapid global dominance of the Delta variant.

13 Jun 2023

What Rosalind Franklin truly contributed to the discovery of DNA’s structure (Nature)

Franklin was no victim in how the DNA double helix was solved. An overlooked letter and an unpublished news article, both written in 1953, reveal that she was an equal player. According to journalist Horace Freeland Judson and Franklin’s biographer, Brenda Maddox, Rosalind Franklin has been reduced to the “wronged heroine” of the double helix. She deserves to be remembered not as the victim of the double helix, but as an equal contributor to the solution of the structure.

13 Jun 2023

Disulfide-Bond-Induced Structural Frustration and Dynamic Disorder in a Peroxiredoxin from MAS NMR

Disulfide bond formation is fundamentally important for protein structure and constitutes a key mechanism by which cells regulate the intracellular oxidation state. Peroxiredoxins (PRDXs) eliminate reactive oxygen species such as hydrogen peroxide through a catalytic cycle of Cys oxidation and reduction. Additionally, upon Cys oxidation PRDXs undergo extensive conformational rearrangements that may underlie their presently structurally poorly defined functions as molecular chaperones. Rearrangements include high molecular-weight oligomerization, the dynamics of which are, however, poorly understood, as is the impact of disulfide bond formation on these properties. Here we show that formation of disulfide bonds along the catalytic cycle induces extensive its time scale dynamics, as monitored by magic-angle spinning NMR of the 216 kDa-large Tsa1 decameric assembly and solution-NMR of a dimeric mutant. We ascribe the conformational dynamics to structural frustration, resulting from conflicts between the disulfide-constrained reduction of mobility and the desire to fulfill other favorable contacts.

13 Jun 2023

How did life begin? One key ingredient is coming into view (Nature)

A Nobel-prizewinning scientist’s team has taken a big step forward in its quest to reconstruct an early-Earth RNA capable of building proteins. Ada Yonath, a structural biologist at the Weizmann Institute of Science in Rehovot, Israel, and her team first conceptualized the ‘protoribosome’ idea nearly two decades ago. The lab’s achievements in the past two years — creating a primitive RNA machine that can link two amino acids together— have created a ripple of excitement.

19 Apr 2023

Mapping the conformational landscape of the stimulatory heterotrimeric G protein (Nature Structural & Molecular Biology)

Heterotrimeric G proteins serve as membrane-associated signaling hubs, in concert with their cognate G-protein-coupled receptors. Fluorine nuclear magnetic resonance spectroscopy was employed to monitor the conformational equilibria of the human stimulatory G-protein alpha subunit (G(s)alpha) alone, in the intact G(s)alpha beta(1)gamma(2) heterotrimer or in complex with membrane-embedded human adenosine A(2A) receptor (A(2A)R).The results reveal a concerted equilibrium that is strongly affected by nucleotide and interactions with the beta gamma subunit, the lipid bilayer and A(2A)R. The alpha 1 helix of G(s)alpha exhibits significant intermediate timescale dynamics. The alpha 4 beta 6 loop and alpha 5 helix undergo membrane/receptor interactions and order-disorder transitions respectively, associated with G-protein activation. The alpha N helix adopts a key functional state that serves as an allosteric conduit between the beta gamma subunit and receptor, while a significant fraction of the ensemble remains tethered to the membrane and receptor upon activation.
Fluorine nuclear magnetic resonance spectroscopy of the stimulatory heterotrimeric G protein reveals a conformational landscape shaped by interactions with nucleotides, the lipid bilayer and a G-protein-coupled receptor.

19 Apr 2023

A quantitative map of nuclear pore assembly reveals two distinct mechanisms (Nature)

Understanding how the nuclear pore complex (NPC) is assembled is of fundamental importance to grasp the mechanisms behind its essential function and understand its role during the evolution of eukaryotes(1-4). There are at least two NPC assembly pathways-one during the exit from mitosis and one during nuclear growth in interphase-but we currently lack a quantitative map of these events. Here we use fluorescence correlation spectroscopy calibrated live imaging of endogenously fluorescently tagged nucleoporins to map the changes in the composition and stoichiometry of seven major modules of the human NPC during its assembly in single dividing cells. This systematic quantitative map reveals that the two assembly pathways have distinct molecular mechanisms, in which the order of addition of two large structural components, the central ring complex and nuclear filaments are inverted. The dynamic stoichiometry data was integrated to create a spatiotemporal model of the NPC assembly pathway and predict the structures of postmitotic NPC assembly intermediates.

19 Apr 2023

Structures and mechanisms of tRNA methylation by METTL1–WDR4 (Nature)

Specific, regulated modification of RNAs is important for proper gene expression(1,2). tRNAs are rich with various chemical modifications that affect their stability and function(3,4). 7-Methylguanosine (m(7)G) at tRNA position 46 is a conserved modification that modulates steady-state tRNA levels to affect cell growth(5,6). The METTL1-WDR4 complex generates m(7)G46 in humans, and dysregulation of METTL1-WDR4 has been linked to brain malformation and multiple cancers(7-22). Here we show how METTL1 and WDR4 cooperate to recognize RNA substrates and catalyse methylation. A crystal structure of METTL1-WDR4 and cryo-electron microscopy structures of METTL1-WDR4-tRNA show that the composite protein surface recognizes the tRNA elbow through shape complementarity. The cryo-electron microscopy structures of METTL1-WDR4-tRNA with S-adenosylmethionine or S-adenosylhomocysteine along with METTL1 crystal structures provide additional insights into the catalytic mechanism by revealing the active site in multiple states. The METTL1 N terminus couples cofactor binding with conformational changes in the tRNA, the catalytic loop and the WDR4 C terminus, acting as the switch to activate m(7)G methylation. Thus, our structural models explain how post-translational modifications of the METTL1 N terminus can regulate methylation. Together, our work elucidates the core and regulatory mechanisms underlying m(7)G modification by METTL1, providing the framework to understand its contribution to biology and disease.

19 Apr 2023

Cryo-EM structure of gas vesicles for buoyancy-controlled motility (Cell)

Gas vesicles are gas-filled nanocompartments that allow a diverse group of bacteria and archaea to control their buoyancy. The molecular basis of their properties and assembly remains unclear. Here, we report the 3.2 Å cryo-EM structure of the gas vesicle shell made from the structural protein GvpA that self-assembles into hollow helical cylinders closed off by cone-shaped tips. Two helical half shells connect through a characteristic arrangement of GvpA monomers, suggesting a mechanism of gas vesicle biogenesis. The fold of GvpA features a corrugated wall structure typical for force-bearing thin-walled cylinders. Small pores enable gas molecules to diffuse across the shell, while the exceptionally hydrophobic interior surface effectively repels water. Comparative structural analysis confirms the evolutionary conservation of gas vesicle assemblies and demonstrates molecular features of shell reinforcement by GvpC. Our findings will further research into gas vesicle biology and facilitate molecular engineering of gas vesicles for ultrasound imaging.

19 Apr 2023

AT-752 targets multiple sites and activities on the Dengue virus replication enzyme NS5 (Antiviral Research)

AT-752 is a guanosine analogue prodrug active against dengue virus (DENV). In infected cells, it is metabolized into 2′-methyl-2′-fluoro guanosine 5′-triphosphate (AT-9010) which inhibits RNA synthesis in acting as a RNA chain terminator. Here we show that AT-9010 has several modes of action on DENV full-length NS5. AT-9010 does not inhibit the primer pppApG synthesis step significantly. However, AT-9010 targets two NS5-associated enzyme activities, the RNA 2′-O-MTase and the RNA-dependent RNA polymerase (RdRp) at its RNA elongation step. Crystal structure and RNA methyltransferase (MTase) activities of the DENV 2 MTase domain in complex with AT-9010 at 1.97 Å resolution shows the latter bound to the GTP/RNA-cap binding site, accounting for the observed inhibition of 2′-O but not N7-methylation activity. AT-9010 is discriminated ∼10 to 14-fold against GTP at the NS5 active site of all four DENV1-4 NS5 RdRps, arguing for significant inhibition through viral RNA synthesis termination. In Huh-7 cells, DENV1-4 are equally sensitive to AT-281, the free base of AT-752 (EC₅₀ ≈ 0.50 μM), suggesting broad spectrum antiviral properties of AT-752 against flaviviruses.