4 Aug 2021

Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants (Nature)

Since the start of the COVID-19 pandemic, SARS-CoV-2 has caused millions of deaths worldwide. Although a number of vaccines have been deployed, the continual evolution of the receptor-binding domain (RBD) of the virus has challenged their efficacy. In particular, the emerging variants B.1.1.7, B.1.351 and P.1 (first detected in the UK, South Africa and Brazil, respectively) have compromised the efficacy of sera from patients who have recovered from COVID-19 and immunotherapies that have received emergency use authorization. One potential alternative to avert viral escape is the use of camelid VHHs (variable heavy chain domains of heavy chain antibody (also known as nanobodies)), which can recognize epitopes that are often inaccessible to conventional antibodies. Here, we isolate anti-RBD nanobodies from llamas and from mice that we engineered to produce VHHs cloned from alpacas, dromedaries and Bactrian camels. We identified two groups of highly neutralizing nanobodies. Group 1 circumvents antigenic drift by recognizing an RBD region that is highly conserved in coronaviruses but rarely targeted by human antibodies. Group 2 is almost exclusively focused to the RBD-ACE2 interface and does not neutralize SARS-CoV-2 variants that carry E484K or N501Y substitutions. However, nanobodies in group 2 retain full neutralization activity against these variants when expressed as homotrimers, and-to our knowledge-rival the most potent antibodies against SARS-CoV-2 that have been produced to date. These findings suggest that multivalent nanobodies overcome SARS-CoV-2 mutations through two separate mechanisms: enhanced avidity for the ACE2-binding domain and recognition of conserved epitopes that are largely inaccessible to human antibodies. Therefore, although new SARS-CoV-2 mutants will continue to emerge, nanobodies represent promising tools to prevent COVID-19 mortality when vaccines are compromised.

12 Jul 2021

Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia (Cell)

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity.

12 Jul 2021

A SARS-CoV-2 neutralizing antibody with extensive Spike binding coverage and modified for optimal therapeutic outcomes (Nature Communications)

COVID-19 pandemic caused by SARS-CoV-2 constitutes a global public health crisis with enormous economic consequences. Monoclonal antibodies against SARS-CoV-2 can provide an important treatment option to fight COVID-19, especially for the most vulnerable populations. In this work, potent antibodies binding to SARS-CoV-2 Spike protein were identified from COVID-19 convalescent patients. Among them, P4A1 interacts directly with and covers majority of the Receptor Binding Motif of the Spike Receptor-Binding Domain, shown by high-resolution complex structure analysis. We further demonstrate the binding and neutralizing activities of P4A1 against wild type and mutant Spike proteins or pseudoviruses. P4A1 was subsequently engineered to reduce the potential risk for Antibody-Dependent Enhancement of infection and to extend its half-life. The engineered antibody exhibits an optimized pharmacokinetic and safety profile, and it results in complete viral clearance in a rhesus monkey model of COVID-19 following a single injection. These data suggest its potential against SARS-CoV-2 related diseases. Antibodies against SARS-CoV-2 S protein can provide a treatment strategy for COVID-19. Here, Guo et al. provide the crystal structure of a SARS-CoV2 neutralizing antibody isolated from a convalescent patient and highlight the therapeutic efficacy in a rhesus monkey model of an engineered version with optimized pharmacokinetic and safety profile.

9 Sep 2022

Selective TnsC recruitment enhances the fidelity of RNA-guided transposition (Nature)

Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD(1), whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs2,3. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins(4). How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous-hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.

11 Jul 2022

De novo design of discrete, stable 310-helix peptide assemblies (Nature)

The α-helix is pre-eminent in structural biology and widely exploited in protein folding, design and engineering. Although other helical peptide conformations do exist near to the α-helical region of conformational space—namely, 3₁₀-helices and π-helices—these occur much less frequently in protein structures. Less favourable internal energies and reduced tendencies to pack into higher-order structures mean that 3₁₀-helices rarely exceed six residues in length in natural proteins, and that they tend not to form normal supersecondary, tertiary or quaternary interactions. Here we show that despite their absence in nature, synthetic peptide assemblies can be built from 3₁₀-helices. We report the rational design, solution-phase characterization and an X-ray crystal structure for water-soluble bundles of 3₁₀-helices with consolidated hydrophobic cores. The design uses six-residue repeats informed by analysing 3₁₀-helical conformations in known protein structures, and incorporates α-aminoisobutyric acid residues. Design iterations reveal a tipping point between α-helical and 3₁₀-helical folding, and identify features required for stabilizing assemblies of 3₁₀-helices. This work provides principles and rules to open opportunities for designing into this hitherto unexplored region of protein-structure space.

11 Jul 2022

Sulfated glycan recognition by carbohydrate sulfatases of the human gut microbiota (Nature Chemical Biology)

Sulfated glycans are ubiquitous nutrient sources for microbial communities that have coevolved with eukaryotic hosts. Bacteria metabolize sulfated glycans by deploying carbohydrate sulfatases that remove sulfate esters. Despite the biological importance of sulfatases, the mechanisms underlying their ability to recognize their glycan substrate remain poorly understood. Here, we use structural biology to determine how sulfatases from the human gut microbiota recognize sulfated glycans. We reveal seven new carbohydrate sulfatase structures spanning four S1 sulfatase subfamilies. Structures of S1_16 and S1_46 represent novel structures of these subfamilies. Structures of S1_11 and S1_15 demonstrate how non-conserved regions of the protein drive specificity toward related but distinct glycan targets. Collectively, these data reveal that carbohydrate sulfatases are highly selective for the glycan component of their substrate. These data provide new approaches for probing sulfated glycan metabolism while revealing the roles carbohydrate sulfatases play in host glycan catabolism.

11 Jul 2022

Cryo-EM structure of a type IV secretion system (Nature)

Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell. It is the primary means by which antibiotic resistance genes spread among bacterial populations. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus—the conjugative type IV secretion system (T4SS)—produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament—the conjugative pilus—that is essential for DNA transfer. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein–protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili.

11 May 2022

CENP-N promotes the compaction of centromeric chromatin (Nature Structural & Molecular Biology)

The histone variant CENP-A is the epigenetic determinant for the centromere, where it is interspersed with canonical H3 to form a specialized chromatin structure that nucleates the kinetochore. How nucleosomes at the centromere arrange into higher order structures is unknown. Here the authors demonstrate that the human CENP-A-interacting protein CENP-N promotes the stacking of CENP-A-containing mononucleosomes and nucleosomal arrays through a previously undefined interaction between the α6 helix of CENP-N with the DNA of a neighboring nucleosome. They describe the cryo-EM structures and biophysical characterization of such CENP-N-mediated nucleosome stacks and nucleosomal arrays and demonstrate that this interaction is responsible for the formation of densely packed chromatin at the centromere in the cell. Their results provide first evidence that CENP-A, together with CENP-N, promotes specific chromatin higher order structure at the centromere.

11 May 2022

True-atomic-resolution insights into the structure and functional role of linear chains and low-barrier hydrogen bonds in proteins (Nature Structural & Molecular Biology)

Hydrogen bonds are fundamental to the structure and function of biological macromolecules and have been explored in detail. The chains of hydrogen bonds (CHBs) and low-barrier hydrogen bonds (LBHBs) were proposed to play essential roles in enzyme catalysis and proton transport. However, high-resolution structural data from CHBs and LBHBs is limited. The challenge is that their ‘visualization’ requires ultrahigh-resolution structures of the ground and functionally important intermediate states to identify proton translocation events and perform their structural assignment. Ttrue-atomic-resolution structures of the light-driven proton pump bacteriorhodopsin, a model in studies of proton transport, show that CHBs and LBHBs not only serve as proton pathways, but also are indispensable for long-range communications, signaling and proton storage in proteins. The complete picture of CHBs and LBHBs discloses their multifunctional roles in providing protein functions and presents a consistent picture of proton transport and storage resolving long-standing debates and controversies.

11 May 2022

What's next for AlphaFold and the AI protein-folding revolution (Nature)

DeepMind software that can predict the 3D shape of proteins is already changing biology. This News Feature published in Nature lists several examples of successful deployment of AlphaFold and RoseTTAFold software and comments their performance.

6 Apr 2022

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase alpha-aminoacrylate intermediate (PNAS)

NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate–dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid–base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate C^β and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.