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Human glycerol channel aquaporin 7 (AQP7) conducts glycerol release from adipocyte and enters the cells in pancreatic islets, muscles, and kidney tubules, and thus regulates glycerol metabolism in those tissues. Compared with other human aquaglyceroporins, AQP7 shows a less conserved “NPA” motif in the center cavity and a pair of aromatic residues at Ar/R selectivity filter. To understand the structural basis for the glycerol conductance, we crystallized the human AQP7 and determined the structure at 3.7 A. A substrate binding pocket was found near the Ar/R filter where a glycerol molecule is bound and stabilized by R229. Glycerol uptake assay on human AQP7 as well as AQP3 and AQP10 demonstrated strong glycerol transportation activities at the physiological condition. The human AQP7 structure, in combination with the molecular dynamics simulation thereon, reveals a fully closed conformation with its permeation pathway strictly confined by the Ar/R filter at the exoplasmic side and the gate at the cytoplasmic side, and the binding of glycerol at the Ar/R filter plays a critical role in controlling the glycerol flux by driving the dislocation of the residues at narrowest parts of glycerol pathway in AQP7.

Green fluorescent proteins (GFPs) have lightened up almost every aspect of biological research including protein sciences. In the field of membrane protein structural biology, GFPs have been used widely to monitor membrane protein localization, expression level, the purification process and yield, and the stability inside the cells and in the test tube. Of particular interest is the fluorescence-detector size-exclusion chromatography-based thermostability assay (FSEC-TS). By simple heating and FSEC, the generally applicable method allows rapid assessment of the thermostability of GFP-fused membrane proteins without purification. Here we describe the experimental details and some typical results for the FSEC-TS method.

Phosphatidylglycerol is a crucial phospholipid found ubiquitously in biological membranes of prokaryotic and eukaryotic cells. The phosphatidylglycerol phosphate (PGP) synthase (PgsA), a membrane-embedded enzyme, catalyzes the primary reaction of phosphatidylglycerol biosynthesis. Mutations in pgsA frequently correlate with daptomycin resistance in Staphylococcus aureus and other prevalent infectious pathogens. Here we report the crystal structures of S. aureus PgsA (SaPgsA) captured at two distinct states of the catalytic process, with lipid substrate (cytidine diphosphate-diacylglycerol, CDP-DAG) or product (PGP) bound to the active site within a trifurcated amphipathic cavity. The hydrophilic head groups of CDP-DAG and PGP occupy two different pockets in the cavity, inducing local conformational changes. An elongated membrane-exposed surface groove accommodates the fatty acyl chains of CDP-DAG/PGP and opens a lateral portal for lipid entry/release. Remarkably, the daptomycin resistance-related mutations mostly cluster around the active site, causing reduction of enzymatic activity. Our results provide detailed mechanistic insights into the dynamic catalytic process of PgsA and structural frameworks beneficial for development of antimicrobial agents targeting PgsA from pathogenic bacteria., Graphical abstract Image 1, Highlights • PgsA uses a trifurcated amphipathic cavity for binding of substrates or products. • Conversion of CDP-DAG to PGP induces local conformational changes in PgsA. • Daptomycin-resistant mutations of PgsA mostly lead to reduced catalytic activity. • A structure-based five-state model is proposed for the synthesis of PGP by PgsA.

International audience; SARS-CoV-2 engages with human cells through the binding of its Spike receptor-binding domain (S-RBD) to the receptor ACE2. Molecular blocking of this engagement represents a proven strategy to treat COVID-19. Here, we report a single-chain antibody (nanobody, DL4) isolated from immunized alpaca with picomolar affinity to RBD. DL4 neutralizes SARS-CoV-2 pseudoviruses with an IC50 of 0.101 mu g mL-1 (6.2 nM). A crystal structure of the DL4-RBD complex at 1.75-angstrom resolution unveils the interaction detail and reveals a direct competition mechanism for DL4's ACE2-blocking and hence neutralizing activity. The structural information allows us to rationally design a mutant with higher potency. Our work adds diversity of neutralizing nanobodies against SARS-CoV-2 and should encourage protein engineering to improve antibody affinities in general.

Polar auxin transport is unique to plants and coordinates their growth and development1,2. The PIN-FORMED (PIN) auxin transporters exhibit highly asymmetrical localizations at the plasma membrane and drive polar auxin transport3,4; however, their structures and transport mechanisms remain largely unknown. Here, we report three inward-facing conformation structures of Arabidopsis thaliana PIN1: the apo state, bound to the natural auxin indole-3-acetic acid (IAA), and in complex with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). The transmembrane domain of PIN1 shares a conserved NhaA fold5. In the substrate-bound structure, IAA is coordinated by both hydrophobic stacking and hydrogen bonding. NPA competes with IAA for the same site at the intracellular pocket, but with a much higher affinity. These findings inform our understanding of the substrate recognition and transport mechanisms of PINs and set up a framework for future research on directional auxin transport, one of the most crucial processes underlying plant development.

An essential step for SARS‐CoV‐2 infection is the attachment to the host cell receptor by its Spike receptor‐binding domain (RBD). Most of the existing RBD‐targeting neutralizing antibodies block the receptor‐binding motif (RBM), a mutable region with the potential to generate neutralization escape mutants. Here, we isolated and structurally characterized a non‐RBM‐targeting monoclonal antibody (FD20) from convalescent patients. FD20 engages the RBD at an epitope distal to the RBM with a K D of 5.6 nM, neutralizes SARS‐CoV‐2 including the current Variants of Concern such as B.1.1.7, B.1.351, P.1, and B.1.617.2 (Delta), displays modest cross‐reactivity against SARS‐CoV, and reduces viral replication in hamsters. The epitope coincides with a predicted “ideal” vulnerability site with high functional and structural constraints. Mutation of the residues of the conserved epitope variably affects FD20‐binding but confers little or no resistance to neutralization. Finally, in vitro mode‐of‐action characterization and negative‐stain electron microscopy suggest a neutralization mechanism by which FD20 destructs the Spike. Our results reveal a conserved vulnerability site in the SARS‐CoV‐2 Spike for the development of potential antiviral drugs., A monoclonal antibody (FD20) from convalescent COVID‐19 patients has been isolated and structurally and biologically characterized. Various SARS‐CoV‐2 strains, including the Alpha, Beta, Gamma, and Delta variants, and naturally occurring epitope mutants, can be neutralized by FD20 with similar potency.

SARS-CoV-2 and its variants continue to threaten public health. The virus recognizes the host cell by attaching its Spike receptor-binding domain (RBD) to the host receptor ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here we report the isolation, and biological and structural characterization of two single-chain antibodies (nanobodies, DL4 and DL28) from RBD-immunized alpaca. Both nanobodies bind Spike with affinities that exceeded the detection limit (picomolar) of the biolayer interferometry assay and neutralize the original SARS-CoV- 2 strain with IC50of 0.086 μg mL-1(DL4) and 0.385 μg mL-1(DL28). DL4 and a more potent, rationally designed mutant, neutralizes the Alpha variant as potently as the original strain but only displays marginal activity against the Beta variant. By contrast, the neutralizing activity of DL28, when in the Fc-fused divalent form, was less affected by the mutations in the Beta variant (IC50of 0.414 μg mL-1for Alpha, 1.060 μg mL-1for Beta). Crystal structure studies reveal that DL4 blocks ACE2-binding by direct competition, while DL28 neutralizes SARS-CoV-2 by an uncommon mechanism through which DL28 distorts the receptor-binding motif in RBD and hence prevents ACE2-binding. Our work provides two neutralizing nanobodies for potential therapeutic development and reveals an uncommon mechanism to design and screen novel neutralizing antibodies against SARS-CoV-2.

SARS-CoV-2, the causative agent of COVID-191, features a receptor-binding domain (RBD) for binding to the host cell ACE2 protein1–6. Neutralizing antibodies that block RBD-ACE2 interaction are candidates for the development of targeted therapeutics7–17. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages in bioavailability, amenability, and production and storage owing to their small sizes and high stability. Here, we report the rapid selection of 99 synthetic nanobodies (sybodies) against RBD by in vitro selection using three libraries. The best sybody, MR3 binds to RBD with high affinity (KD = 1.0 nM) and displays high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.42 μg mL−1). Structural, biochemical, and biological characterization suggests a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency have been generated by structure-based design, biparatopic construction, and divalent engineering. Two divalent forms of MR3 protect hamsters from clinical signs after live virus challenge and a single dose of the Fc-fusion construct of MR3 reduces viral RNA load by 6 Log10. Our results pave the way for the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid development of targeted medical interventions during an outbreak., Here, the authors report the engineering, structural and biological characterization of synthetic nanobodies (sybodies) that display potent therapeutic activity against SARS-CoV-2 infection in animal models via targeting the virus receptor-binding domain.

Breast cancer is a heterogeneous disease. In particular, triple-negative breast cancer (TNBC) comprises various molecular subgroups with unclear identities and currently has few targeted treatment options. Our previous study identified protein C receptor (Procr) as a surface marker on mammary stem cells (MaSCs) located in the basal layer of the normal mammary gland. Given the possible connection of TNBC with basal layer stem cells, we conducted comparative analyses of Procr in breast cancers of mouse and human origin. In mouse mammary tumors, we showed that Procr(+) cells are enriched for cancer stem cells (CSCs) in Wnt1 basal-like tumors, but not in Brca1 basal-like tumors or PyVT luminal tumors. In human cancers, PROCR was robustly expressed in half of TNBC cases. Experiments with patient-derived xenografts (PDXs) revealed that PROCR marks CSCs in this discrete subgroup (referred to as PROCR(+) TNBC). Interfering with the function of PROCR using an inhibitory nanobody reduced the CSC numbers, arrested tumor growth and prevented rapid tumor recurrence. Our data suggest a key role of MaSC in breast tumorigenesis. Moreover, our work indicates that PROCR can be used as a biomarker to stratify TNBC into clinically relevant subgroups and may provide a novel targeted treatment strategy for this clinically important tumor subtype.

Phospholipid biosynthesis begins with the acylation of glycerol 3-phosphate (G3P). In most Gram-positive bacteria including many pathogens, a membrane protein called PlsY is the only acylt...

The 12-transmembrane protein Patched (Ptc1) acts as a negative modulator for Hedgehog (Hh) signaling by depleting sterols in the inner leaflet of the plasma membrane that are required for the activation of downstream regulators in this pathway. The inhibition is alleviated by its proteinaceous ligand Hh. Ptc1 and its co-receptors are locally concentrated in invaginated microdomains known as caveolae for efficient signal transduction. Here, we reconstitute the mouse Ptc1 into lipid nanodiscs and determine its structure in this native-like membrane environment using single-particle cryo-electron microscopy (cryo-EM). The structure is overall similar to the mouse and human Ptc1 structures in amphipol and detergent environment but displays various conformational differences in the transmembrane region. Although most particles show a Ptc1 monomer in the artificial membrane, we observe Ptc1 dimers with distinct interaction patterns and different membrane curvatures. Analyzing the bent membranes reveals that the membrane curvatures are close to that reported in caveolae. We find that a “hand-shake” region rich in hydrophobic and aromatic residues in the ectodomain mediates inter-Ptc1 interactions under different membrane curvatures. We speculate that the plasticity of the hydrophobic interactions is important to accommodate membrane bending. Altogether, our data provide a plausible framework for Ptc1 clustering in the highly curved caveolae membrane.

Green fluorescent proteins (GFPs) are widely used to monitor membrane protein expression, purification, and stability. An ideal reporter should be stable itself and provide high sensitivity and yield. Here, we demonstrate that a coral (Galaxea fascicularis) thermostable GFP (TGP) is by such reasons an improved tag compared to the conventional jellyfish GFPs. TGP faithfully reports membrane protein stability at temperatures near 90 °C (20-min heating). By contrast, the limit for the two popular GFPs is 64 °C and 74 °C. Replacing GFPs with TGP increases yield for all four test membrane proteins in four expression systems. To establish TGP as an affinity tag for membrane protein purification, several high-affinity synthetic nanobodies (sybodies), including a non-competing pair, are generated, and the crystal structure of one complex is solved. Given these advantages, we anticipate that TGP becomes a widely used tool for membrane protein structural studies., In this work, the authors demonstrate that a coral thermostable GFP (TGP) is an improved tag compared to conventional GFPs. In addition to reporting melting point stability at temperatures near 90 °C, its fusion also helps increase expression levels of test membrane proteins. They further generated synthetic nanobodies against TGP to facilitate purification.

SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22-7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8-18. Llama-derived single-domain antibodies (nanobodies, ~15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 μg mL-1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL-1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

The lipid cubic phase (LCP) has been used extensively as a medium for crystallizing membrane proteins. It is an attractive environment in which to perform such studies because it incorporates a lipid bilayer. It is therefore considered a useful and a faithful biomembrane mimetic. Here, we bring together evidence that supports this view. Biophysical characterizations are described demonstrating that the cubic phase is a porous medium into and out of which water-soluble molecules can diffuse for binding to and reaction with reconstituted proteins. The proteins themselves are shown to be functionally reconstituted into and to have full mobility in the bilayered membrane, a prerequisite for LCP crystallogenesis. Spectroscopic methods have been used to characterize the conformation and disposition of proteins in the mesophase. Procedures for performing activity assays on enzymes directly in the cubic phase have been reported. Specific examples described here include a kinase and two transferases, where quantitative kinetics and mechanism-defining measurements were performed directly or via a coupled assay system. Finally, ligand-binding assays are described, where binding to proteins in the mesophase membrane was monitored directly by eye and indirectly by fluorescence quenching, enabling binding constant determinations for targets with affinity values in the micromolar and nanomolar range. These results make a convincing case that the lipid bilayer of the cubic mesophase is an excellent membrane mimetic and a suitable medium in which to perform not only crystallogenesis but also biochemical and biophysical characterizations of membrane proteins.

Here we retrospectively report the successful application of the “host lipid screening” strategy in the in meso crystallization of PlsY, a bacterial glycerol 3-phosphate (G3P) acyltransferase that catalyzes the committed step in phospholipid biosynthesis. Two monoacylglycerols (MAGs) with different chain lengths, namely, the 9.9 MAG (monoolein, nine carbons on either side of the cis-double bond in the acyl chain) and the 7.8 MAG, were the two main host lipids used in this study. The native PlsY crystals growing in 9.9 MAG diffracted to 1.62 A. However, the selenomethioninyl crystals growing in the same host lipid under similar precipitant conditions diffracted poorly (4.3 A). Switching the host lipid to 7.8 MAG dramatically improved crystal quality in both size and diffraction, yielding a 2.0-A data set, with which the structure was solved. Along with low-temperature crystallization, 7.8 MAG was also critical for cocrystallization of PlsY with the unstable lipid substrate, acyl phosphate (acylP). Interest...

The membrane-integral glycerol 3-phosphate (G3P) acyltransferase PlsY catalyses the committed and essential step in bacterial phospholipid biosynthesis by acylation of G3P, forming lysophosphatidic acid. It contains no known acyltransferase motifs, lacks eukaryotic homologs, and uses the unusual acyl-phosphate as acyl donor, as opposed to acyl-CoA or acyl-carrier protein for other acyltransferases. Previous studies have identified several PlsY inhibitors as potential antimicrobials. Here we determine the crystal structure of PlsY at 1.48 Å resolution, revealing a seven-transmembrane helix fold. Four additional substrate- and product-bound structures uncover the atomic details of its relatively inflexible active site. Structure and mutagenesis suggest a different acylation mechanism of ‘substrate-assisted catalysis’ that, unlike other acyltransferases, does not require a proteinaceous catalytic base to complete. The structure data and a high-throughput enzymatic assay developed in this work should prove useful for virtual and experimental screening of inhibitors against this vital bacterial enzyme., The first step in bacterial phospholipid biosynthesis is the acylation of glycerol 3-phosphate to form lysophosphatidic acid. Here, the authors present the high resolution crystal structure of the glycerol 3-phosphate acyltransferase PlsY, a membrane protein and give insights into its catalytical mechanism.

After biosynthesis, bacterial lipopolysaccharides (LPS) are transiently anchored to the outer leaflet of the inner membrane (IM). The ATP-binding cassette (ABC) transporter LptB2FG extracts LPS molecules from the IM and transports them to the outer membrane. Here we report the crystal structure of nucleotide-free LptB2FG from Pseudomonas aeruginosa. The structure reveals that lipopolysaccharide transport proteins LptF and LptG each contain a transmembrane domain (TMD), a periplasmic β-jellyroll-like domain and a coupling helix that interacts with LptB on the cytoplasmic side. The LptF and LptG TMDs form a large outward-facing V-shaped cavity in the IM. Mutational analyses suggest that LPS may enter the central cavity laterally, via the interface of the TMD domains of LptF and LptG, and is expelled into the β-jellyroll-like domains upon ATP binding and hydrolysis by LptB. These studies suggest a mechanism for LPS extraction by LptB2FG that is distinct from those of classical ABC transporters that transport substrates across the IM.

Energy-coupling factor (ECF) transporters are a large family of ATP-binding cassette transporters recently identified in microorganisms. Responsible for micronutrient uptake from the environment, ECF transporters are modular transporters composed of a membrane substrate-binding component EcfS and an ECF module consisting of an integral membrane scaffold component EcfT and two cytoplasmic ATP binding/hydrolysis components EcfA/A'. ECF transporters are classified into groups I and II. Currently, the molecular understanding of group-I ECF transporters is very limited, partly due to a lack of transporter complex structural information. Here, we present structures and structure-based analyses of the group-I cobalt ECF transporter CbiMNQO, whose constituting subunits CbiM/CbiN, CbiQ, and CbiO correspond to the EcfS, EcfT, and EcfA components of group-II ECF transporters, respectively. Through reconstitution of different CbiMNQO subunits and determination of related ATPase and transporter activities, the substrate-binding subunit CbiM was found to stimulate CbiQO's basal ATPase activity. The structure of CbiMQO complex was determined in its inward-open conformation and that of CbiO in β, γ-methyleneadenosine 5'-triphosphate-bound closed conformation. Structure-based analyses revealed interactions between different components, substrate-gating function of the L1 loop of CbiM, and conformational changes of CbiO induced by ATP binding and product release within the CbiMNQO transporter complex. These findings enabled us to propose a working model of the CbiMNQO transporter, in which the transport process requires the rotation or toppling of both CbiQ and CbiM, and CbiN might function in coupling conformational changes between CbiQ and CbiM.

Membrane proteins are generally challenging to work with because of their notorious instability. Protein engineering has been used increasingly to thermostabilize labile membrane proteins such as G-protein coupled receptors for structural and functional studies in recent years. Two major strategies exist. Scanning mutagenesis systematically eliminates destabilizing residues, whereas the consensus approach assembles mutants with the most frequent residues among selected homologs, bridging sequence conservation with stability. Here we applied the consensus concept to stabilize a fungal homolog of the human sterol Δ8-7 isomerase, a 26.4-kDa protein with five transmembrane helices. The isomerase is also called emopamil binding protein (EBP) as it binds this anti-ischemic drug with high affinity. The wild-type had an apparent melting temperature (Tm) of 35.9 °C as measured by the fluorescence-detection size-exclusion chromatography (FSEC)-based thermostability assay. A total of 87 consensus mutations sourced from 22 homologs gained expression level and thermostability, increasing the apparent Tm to 69.9 °C at the cost of partial function loss. Assessing the stability and activity of several systematic chimeric constructs identified a construct with an apparent Tm of 79.8 °C and two regions for function rescue. Further back-mutations of the chimeric construct in the two target regions yielded the final construct with similar apparent activity to the wild-type and an elevated Tm of 88.8 °C, totaling an increase of 52.9 °C. The consensus approach was effective and efficient because it involves fewer constructs compared to scanning mutagenesis. Our results should encourage more use of the consensus strategy for membrane protein thermostabilization.

Human glycerol channel AQP7 conducts glycerol release from adipocyte and entry into the cells in pancreatic islets, muscles and kidney tubule, and thus regulate glycerol metabolism in those tissues. Compared with other human aquaglyceroporins, AQP7 shows a less conserved “NPA” motif in the center cavity, and a pair of aromatic residues at Ar/R selectivity filter. To understand the structural basis for the glycerol conductance, we crystallized the human AQP7 and determined the structure at 3.7 Å. A substrate binding pocket was found near to the Ar/R filter and the bound glycerol molecule stabilized by R229. In vivo functional assay on human AQP7 as well as AQP3 and AQP10 demonstrated strong glycerol transportation activities at physiological condition. The human AQP7 structure reveals a fully closed conformation with its permeation pathway strictly confined by Ar/R filter at the exoplasmic side and the gate at the cytoplasmic side, and the dislocation of the residues at narrowest parts of glycerol pathway in AQP7 play a critical role in controlling the glycerol flux.

The mitochondrial calcium uniporter (MCU) plays a critical role in mitochondrial calcium uptake into the matrix. In metazoans, the uniporter is a tightly regulated multicomponent system, including the pore-forming subunit MCU and several regulators (MICU1, MICU2, and Essential MCU REgulator, EMRE). The calcium-conducting activity of metazoan MCU requires the single-transmembrane protein EMRE. Dictyostelium discoideum (Dd), however, developed a simplified uniporter for which the pore-forming MCU (DdMCU) alone is necessary and sufficient for calcium influx. Here, we report a crystal structure of the N-terminal domain (NTD) of DdMCU at 1.7 Å resolution. The DdMCU-NTD contains four helices and two strands arranged in a fold that is completely different from the known structures of other MCU-NTD homologues. Biochemical and biophysical analyses of DdMCU-NTD in solution indicated that the domain exists as high-order oligomers. Mutagenesis showed that the acidic residues Asp60, Glu72, and Glu74, which appeared to mediate the interface II, as observed in the crystal structure, participated in the self-assembly of DdMCU-NTD. Intriguingly, the oligomeric complex was disrupted in the presence of calcium. We propose that the calcium-triggered dissociation of NTD regulates the channel activity of DdMCU by a yet unknown mechanism.

The mitochondrial calcium uniporter (MCU) plays a critical role in the mitochondrial calcium uptake into the matrix. In metazoans, the uniporter is a tightly regulated multi-component system including the pore-forming subunit MCU and several regulators (MICU1, MICU2, EMRE). The calcium-conducting activity of metazoan MCU requires the single-transmembrane protein EMRE.Dictyostelium discoideum(Dd), however, developed a simplified uniporter for which the pore-forming MCU (DdMCU) alone is necessary and sufficient for calcium influx. Here, we report a crystal structure of the N-terminal domain (NTD) of DdMCU at 1.7 Å resolution. The DdMCU-NTD contains four helices and two strands arranged in a fold that is completely different from the known structures of other MCU-NTD homologs. Biochemical and biophysical analyses of DdMCU-NTD in solution indicated that the domain exists as oligomers, most probably as a pentamer or hexamer. Mutagenesis showed that the acidic residues Asp60, Glu72 and Glu74, which appeared to mediate the parallel interface as observed in the crystal structure, participated in the self-assembly of DdMCU-NTD. Intriguingly, the oligomeric complex readily dissociated to lower-order oligomers in the presence of calcium. We propose that the calcium-triggered dissociation of NTD regulates the channel activity of DdMCU by a yet unknown mechanism.

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and ‘greasy’ site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency., Author summary SARS-CoV-2 relies on the receptor-binding domain (RBD) of its envelope Spike protein to recognize and infect host cells. RBD is therefore an immunological hotspot to generate antibodies for therapeutic and detection purposes. Binding affinity is one of the key characteristics of such antibodies. Here, we report a single-chain antibody (nanobody, ~14 kDa) that binds RBD with nanomolar affinity. The nanobody, dubbed SR31, binds RBD at an epitope distal to the receptor-binding motif (RBM) which is the target of most neutralizing antibodies. SR31 can therefore bind RBD in addition to RBM-binders, and increase their affinity and potency by avidity effects when used as a fusion partner. Compared to other in vitro affinity maturation techniques such as library screening and structure-based design, the fusion strategy offers advantages in speed and simplicity. In addition, SR31, together with RBD-targeting nanobodies recognizing a wide spectrum of epitopes, provides a useful toolkit to probe epitopes of uncharacterized antibodies by competitive binding.

Cholesterol is vital in mammals, but toxic in excess. Consequently, elaborate molecular mechanisms have evolved to maintain this sterol within narrow limits. How cells sense excess cholesterol is an intriguing area of research. Cells sense cholesterol, and other related sterols such as oxysterols or cholesterol synthesis intermediates, and respond to changing levels through several elegant mechanisms of feedback regulation. Cholesterol sensing involves both direct binding of sterols to the homeostatic machinery located in the endoplasmic reticulum (ER), and indirect effects elicited by sterol-dependent alteration of the physical properties of membranes. Here, we examine the mechanisms employed by cells to maintain cholesterol homeostasis.

The successful structure solution of the integral membrane diacylglycerol kinase and the CRISPR-associated endonuclease RNA–DNA complex by native SAD phasing is demonstrated. The structures were solved with a combined low-dose multi-orientation, multi-crystal data-collection strategy., Recent improvements in data-collection strategies have pushed the limits of native SAD (single-wavelength anomalous diffraction) phasing, a method that uses the weak anomalous signal of light elements naturally present in macromolecules. These involve the merging of multiple data sets from either multiple crystals or from a single crystal collected in multiple orientations at a low X-ray dose. Both approaches yield data of high multiplicity while minimizing radiation damage and systematic error, thus ensuring accurate measurements of the anomalous differences. Here, the combined use of these two strategies is described to solve cases of native SAD phasing that were particular challenges: the integral membrane diacylglycerol kinase (DgkA) with a low Bijvoet ratio of 1% and the large 200 kDa complex of the CRISPR-associated endonuclease (Cas9) bound to guide RNA and target DNA crystallized in the low-symmetry space group C2. The optimal native SAD data-collection strategy based on systematic measurements performed on the 266 kDa multiprotein/multiligand tubulin complex is discussed.

Phospholipids constitute a major and essential part of biomembranes and provide mechanical support for membrane proteins. As well, they participate in important cellular activities by interacting with membrane proteins and regulating their functions. In the past decades, enzymes responsible for phospholipid synthesis have been largely identified, purified and characterized, mostly using Escherichia coli and Bacillus subtilis as the model systems. Ever-increasing genome sequencing projects revealed similar enzymes in other microorganisms, as well as new enzymes and pathways for lipids that are traditionally regarded as eukaryotic lipids such as phosphatidylcholine and phosphatidylinositol. Enzymes involved in the phospholipid biosynthesis pathway are mostly associated with the membrane, either peripherally or as an integral part. Structural biology for the pathway had been lacking for these hydrophobic enzymes but are catching up owing to recent technological advances in the membrane protein structural biology field. Here, the biosynthesis pathway of bacterial phospholipids, starting from glycerol 3-phosphate, is summarized. Architecture of the enzymes and mechanisms for substrate binding and catalysis are also illustrated for those whose structural information are available.

The lipid-based bicontinuous cubic mesophase is a nanoporous membrane mimetic with applications in areas that include medicine, personal care products, foods, and the basic sciences. An application of particular note is as a medium in which to grow crystals of membrane proteins for structure determination by X-ray crystallography. At least two variations of the mesophase exist. One is the highly viscous cubic phase, known as the lipid cubic phase (LCP), which has well-developed long-range order. The other, the so-called sponge phase, is considerably more fluid and lacks long-range order. Both phase types have been shown to be amenable for growing microcrystals of membrane proteins and for use as a delivery medium to shuttle protein crystals to an X-ray free-electron laser beam for serial femtosecond crystallography. Here, we provide background on the physicochemical properties of these mesophases and how they function to grow microcrystals of membrane proteins. Protocols implemented for the generation and use of nanoliter volumes of mesophase of suitably high microcrystal density required for serial femtosecond crystallography are described. Prospects for future uses of lipid mesophases in the serial femtosecond crystallography arena are summarized.

Photoacoustic (PA) spectroscopy technology has proved to be effective in detection of SF6 decomposition products. But in the real application environment, the stability of the PA spectroscopy device is strongly affected by ambient temperature. In this paper, we took CO as an example to analyze the impact mechanism of temperature and the temperature correction formula of the response factor of carbon monoxide (PA signal corresponding to unit concentration gas) was derived. The real temperature properties of SF6 and CO were studied using a non-resonant PA spectroscopy device designed by our team. The study indicates that the PA signals of SF6 and CO are inversely proportional to temperature, which coincides with the theoretical derivation. A temperature correction method was proposed based on theoretical derivation combined with experimental study and this method was tested and validated. The results show that the relative errors of calculated concentrations of CO acquired by the correction method compared to the actual concentrations are all within 10%, which meets the detection requirements of PA spectroscopy for SF6 decomposition products in gas insulated switchgear. The correction method has provided an effective way for studying the temperature correction in PA detection for SF6 decomposition products in GIS.

Under partial discharge (PD), a gas-insulated switchgear (GIS) will produce SF6 decomposition products. If the concentrations of these products are higher than the standard values, this indicates that the GIS equipment is faulty. Photoacoustic spectroscopy techniques show a high sensitivity and good stability and, furthermore, they can enable on-line monitoring, making them widely used in the detection of SF6 decomposition products in GIS equipment. The basic principles of photoacoustic spectroscopy were introduced and a set of devices for the online detection of SF6 decomposition based on non-resonant photoacoustics was designed and realized. According to the infrared spectral characteristics of the target gases, the appropriate narrowband filters and infrared radiation light source were selected. This detection system is able to continuously detect CO, SO2 and CF4. Calibration experiments with different concentrations of CO, SO2 and CF4 were carried out and the results show that the photoacoustic signal and concentration have a perfect linear relationship and the detection limits for CO, SO2 and CF4 are 5.9116, 8.2824 and 5.5226 ppm. Relative errors of the single measurements of CO, SO2 and CF4 are all less than 10% and the system exhibits excellent stability in a continuous 12 hour measurement.