An extended conformation of SARS-CoV-2 main protease reveals allosteric targets.

SignificanceThe coronavirus main protease (Mpro) is required for viral replication. Here, we obtained the extended conformation of the native monomer of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Mpro by trapping it with nanobodies and found that the catalytic domain and the helix domain dissociate, revealing allosteric targets. Another monomeric state is termed compact conformation and is similar to one protomer of the dimeric form. We designed a Nanoluc Binary Techonology (NanoBiT)-based high-throughput allosteric inhibitor assay based on structural conformational change. Our results provide insight into the maturation, dimerization, and catalysis of the coronavirus Mpro and pave a way to develop an anticoronaviral drug through targeting the maturation process to inhibit the autocleavage of Mpro.

A procedure for producing an anti-AXL nanobody in E. coli.

As one of the receptors of the TAM family, AXL plays a vital role in stem cell maintenance, angiogenesis, immune escape of viruses and drug resistance against tumors. In this study, the truncated extracellular segment containing two immunoglobulin-like domains of human AXL (AXL-IG), which has been confirmed to bind growth arrest specific 6 (GAS6) by structural studies [1], was expressed in a prokaryotic expression system and then purified. Immunizing camelid with the purified AXL-IG as antigen could lead to the production of unique nanobodies composed of only variable domain of heavy chain of heavy-chain antibody (VHH), which are around 15 kD and stable. We screened out a nanobody A-LY01 specific binding to AXL-IG. We further determined the affinity of A-LY01 to AXL-IG and revealed that A-LY01 could specifically recognize full-length AXL on the surface of HEK 293T/17 cells. Our study provides appropriate support for the development of diagnostic reagents and antibody therapeutics targeting AXL.

Development of a nanobody-based immunoassay for the sensitive detection of fibrinogen-like protein 1.

Immune checkpoint inhibition is an important strategy in cancer therapy. Blockade of CTLA-4 and PD-1/PD-L1 is well developed in clinical practice. In the last few years, LAG-3 has received much interest as an emerging novel target in immunotherapy. It was recently reported that FGL1 is a major ligand of LAG-3, which is normally secreted by the liver but is upregulated in several human cancers. FGL1 is a crucial biomarker and target for cancer immunotherapy. As the efficacy of immunotherapy is limited to specific types of patients, the subset of patients needs to be selected appropriately to receive precise treatment according to different biomarkers. To date, there is no test to accurately assess FGL1 expression levels. Nanobodies have some outstanding features, such as high stability, solubility and affinity for diagnostic and therapeutic applications. Here, we report the development and validation of a rapid, sensitive, and cost-effective nanobody-based immunoassay for the detection of FGL1 in human serum. In this study, human FGL1 recombinant protein was expressed and purified for the first time as an immunized antigen. Then, we constructed a nanobody phage display library and screened several nanobodies that bind FGL1 with high affinity. We selected two nanobodies targeting different epitopes of FGL1, one as a capture and the other conjugated with HRP as a probe. The double nanobody-based sandwich ELISA to detect the concentration of FGL1 showed a good response relationship in the range of 15.625-2000 ng/mL, and the recoveries from the spiked sample were in the range of 78% and 100%. This assay could be used as a potential approach for evaluating FGL1 expression for patient stratification and for predicting the therapeutic efficacy of targeting the LAG3/FGL1 axis.

Charge density waves in individual nanoribbons of orthorhombic-TaS3.

Orthorhombic-TaS3 is a quasi-1D material that undergoes a Peierls' transition to become a charge density wave conductor at low temperatures. Electrical transport measurements of individual single-crystalline TaS3 nanoribbons prepared by a novel bottom-up method from elemental precursors indicate a depression of the Peierls' ordering temperature to 205 K, broadening of the electric-field-induced depinning of the charge density wave below the Peierls' transition temperature, and a greatly increased threshold voltage for nucleation of charge density wave dislocations posited to be a result of surface confinement and finite size effects. Single-nanoribbon measurements of broad-band noise indicate discrete phase slip events near the depinning threshold. Three distinct regimes are identified with the normalized noise spectrum showing a distinctive maxima near the threshold voltage for depinning of the charge density wave, corresponding to sampling of different metastable states that balance ordered and sliding charge density waves.

Covalently bridging graphene edges for improving mechanical and electrical properties of fibers.

Assembling graphene sheets into macroscopic fibers with graphitic layers uniaxially aligned along the fiber axis is of both fundamental and technological importance. However, the optimal performance of graphene-based fibers has been far lower than what is expected based on the properties of individual graphene. Here we show that both mechanical properties and electrical conductivity of graphene-based fibers can be significantly improved if bridges are created between graphene edges through covalent conjugating aromatic amide bonds. The improved electrical conductivity is likely due to extended electron conjugation over the aromatic amide bridged graphene sheets. The larger sheets also result in improved π-π stacking, which, along with the robust aromatic amide linkage, provides high mechanical strength. In our experiments, graphene edges were bridged using the established wet-spinning technique in the presence of an aromatic amine linker, which selectively reacts to carboxyl groups at the graphene edge sites. This technique is already industrial and can be easily upscaled. Our methodology thus paves the way to the fabrication of high-performance macroscopic graphene fibers under optimal techno-economic and ecological conditions.

Discovery of quantum phases in the Shastry-Sutherland compound SrCu2(BO3)2 under extreme conditions of field and pressure.

The 2-dimensional layered oxide material SrCu2(BO3)2, long studied as a realization of the Shastry-Sutherland spin topology, exhibits a range of intriguing physics as a function of both hydrostatic pressure and magnetic field, with a still debated intermediate plaquette phase appearing at approximately 20 kbar and a possible deconfined critical point at higher pressure. Here, we employ a tunnel diode oscillator (TDO) technique to probe the behavior in the combined extreme conditions of high pressure, high magnetic field, and low temperature. We reveal an extensive phase space consisting of multiple magnetic analogs of the elusive supersolid phase and a magnetization plateau. In particular, a 10 × 2 supersolid and a 1/5 plateau, identified by infinite Projected Entangled Pair States (iPEPS) calculations, are found to rely on the presence of both magnetic and non-magnetic particles in the sea of dimer singlets. These states are best understood as descendants of the full-plaquette phase, the leading candidate for the intermediate phase of SrCu2(BO3)2.

Structural basis of nanobodies neutralizing SARS-CoV-2 variants.

Because of the evolutionary variants of SARS-CoV-2, development of broad-spectrum neutralizing antibodies resilient to virus escape is urgently needed. We identified a group of high-affinity nanobodies from camels immunized with receptor-binding domain (RBD) of SARS-CoV-2 spike protein and resolved the structures of two non-competing nanobodies (NB1A7 and NB1B11) in complex with RBD using X-ray crystallography. The structures show that NB1A7 targets the highly conserved cryptic epitope shared by SARS-CoV-2 variants and some other coronaviruses and blocks ACE2 receptor attachment of the spike protein, and NB1B11 epitope overlaps with the contacting surface of ACE2 and is different from the binding site of NB1A7. These two nanobodies were covalently linked into multivalent and bi-paratopic formats, which significantly improved the avidity and neutralization potency and may further inhibit viral escape. The results contribute to the structure-guided design of antibodies against future variants of SARS-CoV-2 virus to combat coronavirus epidemics and pandemics.

Magnetic field reveals vanishing Hall response in the normal state of stripe-ordered cuprates.

The origin of the weak insulating behavior of the resistivity, i.e. [Formula: see text], revealed when magnetic fields (H) suppress superconductivity in underdoped cuprates has been a longtime mystery. Surprisingly, the high-field behavior of the resistivity observed recently in charge- and spin-stripe-ordered La-214 cuprates suggests a metallic, as opposed to insulating, high-field normal state. Here we report the vanishing of the Hall coefficient in this field-revealed normal state for all [Formula: see text], where [Formula: see text] is the zero-field superconducting transition temperature. Our measurements demonstrate that this is a robust fundamental property of the normal state of cuprates with intertwined orders, exhibited in the previously unexplored regime of T and H. The behavior of the high-field Hall coefficient is fundamentally different from that in other cuprates such as YBa2Cu3O6+x and YBa2Cu4O8, and may imply an approximate particle-hole symmetry that is unique to stripe-ordered cuprates. Our results highlight the important role of the competing orders in determining the normal state of cuprates.

Structural insights into the activation of human calcium-sensing receptor.

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that maintains Ca2+ homeostasis in serum. Here, we present the cryo-electron microscopy structures of the CaSR in the inactive and agonist+PAM bound states. Complemented with previously reported structures of CaSR, we show that in addition to the full inactive and active states, there are multiple intermediate states during the activation of CaSR. We used a negative allosteric nanobody to stabilize the CaSR in the fully inactive state and found a new binding site for Ca2+ ion that acts as a composite agonist with L-amino acid to stabilize the closure of active Venus flytraps. Our data show that agonist binding leads to compaction of the dimer, proximity of the cysteine-rich domains, large-scale transitions of seven-transmembrane domains, and inter- and intrasubunit conformational changes of seven-transmembrane domains to accommodate downstream transducers. Our results reveal the structural basis for activation mechanisms of CaSR and clarify the mode of action of Ca2+ ions and L-amino acid leading to the activation of the receptor.

Vortex phase diagram and the normal state of cuprates with charge and spin orders.

The phase diagram of underdoped cuprates in a magnetic field (H) is key to understanding the anomalous normal state of these high-temperature superconductors. However, the upper critical field (H c2), the extent of superconducting (SC) phase with vortices, and the role of charge orders at high H remain controversial. Here we study stripe-ordered La-214, i.e., cuprates in which charge orders are most pronounced and zero-field SC transition temperatures T c 0 are lowest. This enables us to explore the vortex phases in a previously inaccessible energy scale window. By combining linear and nonlinear transport techniques sensitive to vortex matter, we determine the T - H phase diagram, directly detect H c2, and reveal novel properties of the high-field ground state. Our results demonstrate that quantum fluctuations and disorder play a key role as T → 0, while the high-field ground state is likely a metal, not an insulator, due to the presence of stripes.

Pair density wave at high magnetic fields in cuprates with charge and spin orders.

In underdoped cuprates, the interplay of the pseudogap, superconductivity, and charge and spin ordering can give rise to exotic quantum states, including the pair density wave (PDW), in which the superconducting (SC) order parameter is oscillatory in space. However, the evidence for a PDW state remains inconclusive and its broader relevance to cuprate physics is an open question. To test the interlayer frustration, the crucial component of the PDW picture, we perform transport measurements on charge- and spin-stripe-ordered La1.7Eu0.2Sr0.1CuO4 and La1.48Nd0.4Sr0.12CuO4 in perpendicular magnetic fields (H⊥), and also with an additional field applied parallel to CuO2 layers (H∥). We detect several phenomena predicted to arise from the existence of a PDW, including an enhancement of interlayer SC phase coherence with increasing H∥. These data also provide much-needed transport signatures of the PDW in the regime where superconductivity is destroyed by quantum phase fluctuations.

Emergent bound states and impurity pairs in chemically doped Shastry-Sutherland system.

Impurities often play a defining role in the ground states of frustrated quantum magnets. Studies of their effects are crucial in understanding of the phase diagram in these materials. SrCu2(BO3)2, an experimental realization of the Shastry-Sutherland (SS) lattice, provides a unique model system for such studies using both experimental and numerical approaches. Here we report effects of impurities on the crystals of bound states, and doping-induced emergent ground states in Mg-doped SrCu2(BO3)2, which remain stable in high magnetic fields. Using four complementary magnetometry techniques and theoretical simulations, a rich impurity-induced phenomenology at high fields is discovered. The results demonstrate a rare example in which even a small doping concentration interacts strongly with both triplets and bound states of triplets, and thus plays a significant role in the magnetization process even at high magnetic fields. Our findings provide insights into the study of impurity effects in geometrically frustrated quantum magnets.