Journal: Nature communications

Article Title: Cryo-EM structures of the membrane repair protein dysferlin.

doi: 10.1038/s41467-024-53773-6

Figure Lengend Snippet: Fig. 3 | Key interactions in the dysferlin monomer. a Two views (front, back) of the pentameric ring formed by the C2B, C2E, C2D, C2C, and composite C2 domain and stabilized by the C2D-C2E linker. Individual domains are color-coded. b Schematic representation of the pentameric ring. Color code according to panel a. c Residue-level interactions at interdomain interfaces in the pentameric ring. d Residue-level interactions of the FerI with the C2B, C2C, and C2E. Color code according to panel a. The electrostatic potential distribution is shown for the C2B, C2C, and C2E (left). e The C2E-C2F (grey) and C2F-C2G (grey) linkers connect the respective domains. The C2E insert interacts with the C2E (yellow), C2F (blue), and

Article Snippet: The codon-optimized dysferlin cDNA (Addgene plasmid # 67878, a gift from Matthew Hirsch)60 was amplified and inserted upstream of a coding sequence for a PreScission protease cleavage site and two strep II tags into a pFastBac1 vector via restriction-fee cloning.

Techniques: Residue

Journal: Nature Communications

Article Title: Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

doi: 10.1038/s41467-021-27611-y

Figure Lengend Snippet: a ELISA screen for identification of potential SARS-CoV-2 RBD binders. Negative control wells containing expression media, and positive control wells containing VNAR E06 (anti-serum albumin) and rabbit monoclonal CR3022-RB (anti-SARS-CoV-2 Spike) are indicated. b Primary screen for identification of neutralizing VNAR domains. Concentration-dependent neutralization of pseudotyped SARS-CoV-2 (black) or SARS-CoV-1 (red) in HEK293T cells transiently expressing ACE2. Data represents mean ± s.e.m. relative luminescence units (RLUs) from n = 3 independent biological experiments. c Left , rank-ordered IC 50 values for neutralizing VNARs from panel ( b ). VNARs with IC 50 < 10 nM (dashed line) were selected for further characterization. Upper right, depiction of primary sequences of selected VNARs, relative length of complementarity determining regions (CDR1, CDR3) and location of cysteine residues (teal) are shown. d Phylogenetic tree of selected virus taxa, divergent lineages of betacoronaviruses are shown. Glycoproteins encoded by the indicated viruses (*) were used to generate pseudoviruses. e Heatmap summarizing IC 50 values for neutralization of the indicated pseudovirus with the indicated VNAR antibody. Values are derived from experiments described in ( f ). f Secondary validation of selected neutralizing VNAR domains. Concentration-dependent neutralization of viral particles pseudotyped with glycoproteins natively encoded by either SARS-CoV-2 (black), SARS-CoV-1 (red), WIV1-CoV (blue). MERS-CoV (green), or VSV (purple) in Calu-3 cells. Cell viability was also assessed in the presence of increasing concentrations of VNARs (yellow). Data represents mean ± s.e.m. RLU values from n = 3 independent biological experiments. g Concentration-dependent neutralization of replication-competent authentic SARS-CoV-2, strain USA_WA1/2020 in Vero E6 cells. Data represents mean ± s.e.m. RFU values from n = 3 independent biological experiments.

Article Snippet: Plasmids used for generating pseudovirus stocks were sourced as follows: plasmid encoding an Env -defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.R-E − ) , was obtained through the NIH AIDS Reagent Program; plasmids encoding SARS-CoV-1 and SARS-CoV-2 Spike were from Fang Li (Addgene plasmid #145031 and #145032, respectively) ; WIV1-CoV Spike was from Alejandro Balazs (Addgene plasmid #164439) ; MERS-CoV Spike was from Sino Biological (VG40069-NF); and VSV-G was from Bob Weinberg (Addgene plasmid #8454).

Techniques: Enzyme-linked Immunosorbent Assay, Negative Control, Expressing, Positive Control, Concentration Assay, Neutralization, Derivative Assay

Journal: Nature Communications

Article Title: Mechanisms of SARS-CoV-2 neutralization by shark variable new antigen receptors elucidated through X-ray crystallography

doi: 10.1038/s41467-021-27611-y

Figure Lengend Snippet: a Sequence alignment between SARS-CoV-1, SARS-CoV-2, and the MERS RBDs. Residues different from SARS-CoV-2 are highlighted in red and the interaction interface for VNAR-3B4 is marked with a blue box. Bolded letters indicate residues critical for the interaction between the RBD and 3B4 with arrows indicating the residues that form a backbone beta-sheet. The sequence alignment is numbered above according to SARS-CoV-2. b Surface representations of SARS-CoV-1 (pink, above) and MERS (green, below) with variant residues colored red. The ACE2 binding interface is highlighted in purple for SARS-CoV-1 and the DPP4 binding interface in orange for MERS. The homology-modeled interaction interface for 3B4 is colored blue for both structures. c Zoomed in view of the modeled interaction interface between 3B4 (blue) and SARS-CoV-1 (pink, above) and MERS (green, below), with 3B4 colored blue in both pictures. Interacting residues are highlighted as in Fig. , showing the backbone interactions in black dashes and sidechain to backbone or sidechain to sidechain interactions shown as yellow dashes. Insets show the orientation of the zoomed in view. d Overlays of the 3B4 interface from modeled RBDs and their matching RBDs obtained by x-ray crystallography. The panel above shows the modeled SARS-CoV-1 RBD, colored pink, aligned with the crystal structure of SARS-CoV-1 RBD bound to ACE2 (PDB id: 2AJF), colored magenta. The panel below shows the modeled MERS RBD, colored light green, aligned with the crystal structure of MERS RBD bound to DPP4 (PDB id: 4L72), colored dark green.

Article Snippet: Plasmids used for generating pseudovirus stocks were sourced as follows: plasmid encoding an Env -defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.R-E − ) , was obtained through the NIH AIDS Reagent Program; plasmids encoding SARS-CoV-1 and SARS-CoV-2 Spike were from Fang Li (Addgene plasmid #145031 and #145032, respectively) ; WIV1-CoV Spike was from Alejandro Balazs (Addgene plasmid #164439) ; MERS-CoV Spike was from Sino Biological (VG40069-NF); and VSV-G was from Bob Weinberg (Addgene plasmid #8454).

Techniques: Sequencing, Variant Assay, Binding Assay

Journal: Journal of Cell Science

Article Title: Visualizing endogenous Rho activity with an improved localization-based, genetically encoded biosensor

doi: 10.1242/jcs.258823

Figure Lengend Snippet: The optimized dimericTomato-2xrGBD Rho sensor has the best location efficiency. (A) Spinning disk still images of a HeLa cells expressing the CMVdel-mNeonGreen-1xrGBD Rho sensor (upper panel) or CMVdel-dimericTomato-2xrGBD Rho sensor (lower panel) and H1R (not shown), which were stimulated with 100 μM histamine after 150 s and 10 μM pyrilamine after 350 s. The images match with the perturbation that is indicated for the plot in C. Times are min:s from the start of the recording. Scale bars: 25 µm. (B) Change in cytosolic intensity for CMV-mNeonGreen, CMV-eGFP-1xrGBD, CMVdel-3xmNeonGreen-1xrGBD, CMVdel-mNeonGreen-1xrGBD, -2xrGBD and -3xrGBD, and CMVdel-dimericTomato-1xrGBD and -2xrGBD (schematics on left) in HeLa cells expressing H1R, upon stimulation with 100 μM histamine. Each dot represents an individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The gray dashed line indicates no change in cytosolic intensity. The data is from two biological replicates based on two independent transfections. The number of cells per condition is: 3xmNG-1xrGBD=27, dT-1xrGBD=32, dT-2xrGBD=33, eGFP-1xrGBD=40, mNG=39, mNG-1xrGBD=28, mNG-2xrGBD=34, mNG-3xrGBD=26. (C) Time traces of the change in cytosolic intensity for the displayed cells for the mNeonGreen-1xrGBD sensor in gray and for the dimericTomato-2xrGBD sensor in black. mNG, mNeonGreen; dT, dimericTomato; rGBD, rhotekin G protein binding domain.

Article Snippet: The following plasmids, generated in this study, are available on Addgene ( http://www.addgene.org/ ): 129625 , dTomato-2xrGBD (C1 vector); 176098 , dimericTomato-2xrGBD (pLV vector); 129624 , mNeongreen-2xrGBD; 176091 , mNeonGreen-3xrGBD; 129633 , mNeonGreen-aGBD (anillin); 129634 , mNeonGreen-pGBD (PKN1 codon optimized); 176094 , H2A-mTurquoise2-CDC42-G12V-ΔCaaX; 176095 , H2A-mTurquoise2-RAC1-G12V-ΔCaaX; and 176097 , H2A-mTurquoise2-RHOA-G14V-ΔCaaX.

Techniques: Expressing, Transfection, Protein Binding

Journal: Journal of Cell Science

Article Title: Visualizing endogenous Rho activity with an improved localization-based, genetically encoded biosensor

doi: 10.1242/jcs.258823

Figure Lengend Snippet: The GBDs of anillin and PKN1 are not suitable for a relocation Rho sensor. (A) Change in cytosolic intensity for CMVdel-mNeonGreen-1xpGBD, -2xpGBD and -3xpGBD, and CMVdel-dimericTomato-2xpGBD co expressed with H1R in HeLa cells upon stimulation with 100 μM histamine. The dashed line represents no change in cytosolic intensity. Each dot represents an individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The number of samples per condition is: dT-2xpGBD=16, mNG-1xpGBD=24, mNG-2xpGBD=28, mNG-3xpGBD=14. (B) Spinning disk images showing colocalization of H2A-mTurquoise2-RhoAG14V-ΔCaaX or control H2A-mTurquoise2 with dimericTomato-2xpGBD in HeLa cells. Scale bars: 20 µm. RhoA binding, represented by the ratio of sensor intensity in the nucleus to cytosol in H2A-mTurquoise2-RhoAG14V-ΔCaaX expressing HeLa cells. The dashed line indicates a ratio of one. Each dot represents an individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The number of cells from a single replicate per condition is: H2A-dT-2xpGBD=19, H2A-RhoA-dT2xpGBD=16. (C) Change in cytosolic intensity for CMVdel-mNeonGreen-1xaGBD, -2xaGBD and -3xaGBD, CMVdel-dimericTomato-1xaGBD and eGFP-anillin(AHD+PH) coexpressed with H1R in HeLa cells upon stimulation with 100 μM histamine. The dashed line represents no change in cytosolic intensity. Each dot represents an individual cell. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The number of cells per condition is: dT-1xaGBD=14, eGFP-AHD+PH=27, mNG-1xaGBD=17, mNG-2xaGBD=10, mNG-3xaGBD=13. (D) Spinning disk images showing colocalization of H2A-mTurquoise2-RhoAG14V-ΔCaaX or control H2A-mTurquoise2 with dimericTomato-1xaGBD and mScarlet-I-anillin(AHD+PH) in HeLa cells. Scale bars: 20 µm. RhoA binding, represented by the ratio of sensor intensity in the nucleus to cytosol in H2A-mTurquoise2-RhoAG14V-ΔCaaX-expressing HeLa cells. The dashed line indicates a ratio of one. The median of the data is shown as a black circle and the 95% confidence interval for each median, determined by bootstrapping, is indicated by the bar. The number of cells from a single replicate per condition is: H2A-aGBD=12, H2A-RhoA-aGBD=10, H2A-RhoA-AHD+PH=13. (E) Amino acid sequence alignment for aGBD, rGBD and pGBD from MUSCLE and depicted with the clustalX color code, where green is polar, blue is hydrophobic, purple is negative charge, red is positive charge, yellow is prolines, orange is glycines, cyan is aromatic and white is unconserved. (F) Crystal structures of PKN1 G-protein binding domain (purple) and anillin G-protein binding domain (yellow), bound to RhoA GTP (gray). On the left, a structural alignment of PKN1 and anillin by their G-protein binding domains. On the right, a structural alignment of the bound RhoA molecules, showing the two binding positions at the RhoA molecule (PDB: anillin, 4XOI ; PKN1, 1CXZ ).

Article Snippet: The following plasmids, generated in this study, are available on Addgene ( http://www.addgene.org/ ): 129625 , dTomato-2xrGBD (C1 vector); 176098 , dimericTomato-2xrGBD (pLV vector); 129624 , mNeongreen-2xrGBD; 176091 , mNeonGreen-3xrGBD; 129633 , mNeonGreen-aGBD (anillin); 129634 , mNeonGreen-pGBD (PKN1 codon optimized); 176094 , H2A-mTurquoise2-CDC42-G12V-ΔCaaX; 176095 , H2A-mTurquoise2-RAC1-G12V-ΔCaaX; and 176097 , H2A-mTurquoise2-RHOA-G14V-ΔCaaX.

Techniques: Binding Assay, Expressing, Sequencing, Protein Binding