Journal: Scientific Reports

Article Title: High affinity nanobodies block SARS-CoV-2 spike receptor binding domain interaction with human angiotensin converting enzyme

doi: 10.1038/s41598-020-79036-0

Figure Lengend Snippet: Protein sequences for novel nanobodies that bind to the SARS-CoV-2 spike protein receptor binding domain. Single letter amino acid codes. Clustal Omega algorithm used for alignment. Blue highlights indicate sequence diversity with NIH-CoVnb-112, highlighted in gray, set as the reference sequence for comparison. For comparison, seven previously reported nanobody sequences have clearly distinct sequences: Ty1 , VHH72 , H11-D4 , MR3 , Sb#14 , Sb23 , and W25UACh and possess shorter CDR3 domains (represented in NIH-CoVnb-112 by amino acids 99–120).

Article Snippet: Sequences were trimmed to include only the VHH coding region and the protein coding sequences aligned using the Clustal Omega algorithm included in SnapGene software (GSL Biotech LLC).

Techniques: Binding Assay, Sequencing, Comparison

Journal: Scientific Reports

Article Title: Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation

doi: 10.1038/s41598-019-48751-8

Figure Lengend Snippet: Structural features of and interaction model for Grp94 and myocilin. ( A ) Model of Grp94 highlighting its structural domains: Pre-N subdomain, cyan; N-terminal ATP-binding domain (N), light blue; middle domain (M), blue; C-terminal dimerization domain (C), purple. The second monomer of the obligate homodimer is shaded gray for clarity. ( B ) Schematic of myocilin structural organization emphasizing the C-terminal olfactomedin (OLF) domain in green. Colored spheres represent calcium (orange) and potassium (magenta) ions. Models are not drawn to scale. Models are from structures with PDB-ID codes 5ULS (Grp94) and 4WXQ, 5VR2 (myocilin). ( C ) Model of Grp94 involvement in myocilin aggregation and glaucoma-associated cellular toxicity. Grp94 is recruited by misfolded, aggregating mutant myocilin via the ERAD system, but counterproductively facilitates aggregation and preserves toxic aggregates; preventing the Grp94/myocilin interaction is a viable method of rescuing cells from cytotoxicity via alternative clearance mechanisms. Myoc = myocilin.

Article Snippet: Grp94 MC protein domain construct gene (residues 336–765, Canis lupus familiaris origin, 98.4% identity to human sequence for Grp94 MC , 97.9% identity overall based on Clustal Omega alignment ) was synthesized and codon-optimized for E . coli expression by ATUM and cloned into a pMAL-c5X vector (New England Biolabs) so that it contained a TEV-cleavable N-terminal maltose binding protein (MBP), and a Factor Xa-cleavable C-terminus 6xHis tag.

Techniques: Binding Assay, Mutagenesis

Journal: Scientific Reports

Article Title: Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation

doi: 10.1038/s41598-019-48751-8

Figure Lengend Snippet: The N-terminal domain of Grp94 is responsible for the aberrant Grp94/OLF protein-protein interaction. ( A ) Grp94 N enhances the rate of OLF aggregation, whereas Grp94 MC appears to stabilize OLF against aggregation as indicated by ThT fluorescence. Results represent the average of 20 (OLF + Grp94 N ), 12 (OLF + Grp94 NM ), and 12 (OLF + Grp94 MC ) replicates from at least 2 biological replicates. The ^ symbol represents data presented previously ; ***(p < 0.0001) represents statistically significant differences relative to OLF at 24 hours. ( B ) The Grp94 N-terminal domain and OLF co-aggregate over the course of the aggregation assay in ( A ). S = supernatant, W = wash, and P = pellet/aggregate. See Fig. for full four-day kinetics assay data, and Fig. for additional co-aggregation SDS-PAGE gels for the remaining domain constructs.

Article Snippet: Grp94 MC protein domain construct gene (residues 336–765, Canis lupus familiaris origin, 98.4% identity to human sequence for Grp94 MC , 97.9% identity overall based on Clustal Omega alignment ) was synthesized and codon-optimized for E . coli expression by ATUM and cloned into a pMAL-c5X vector (New England Biolabs) so that it contained a TEV-cleavable N-terminal maltose binding protein (MBP), and a Factor Xa-cleavable C-terminus 6xHis tag.

Techniques: Fluorescence, SDS Page, Construct

Journal: Scientific Reports

Article Title: Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation

doi: 10.1038/s41598-019-48751-8

Figure Lengend Snippet: Impact of Grp94 domains on OLF aggregation.

Article Snippet: Grp94 MC protein domain construct gene (residues 336–765, Canis lupus familiaris origin, 98.4% identity to human sequence for Grp94 MC , 97.9% identity overall based on Clustal Omega alignment ) was synthesized and codon-optimized for E . coli expression by ATUM and cloned into a pMAL-c5X vector (New England Biolabs) so that it contained a TEV-cleavable N-terminal maltose binding protein (MBP), and a Factor Xa-cleavable C-terminus 6xHis tag.

Techniques:

Journal: Scientific Reports

Article Title: Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation

doi: 10.1038/s41598-019-48751-8

Figure Lengend Snippet: Mapping the Grp94/OLF protein-protein interaction interface. ( A ) Coomassie blue (left) and dansyl fluorescence (right) visualization of crosslinking reaction products (arrows) as seen by SDS-PAGE for the coupling of Grp94 NM with OLF. Dashed line distinguishes different visualizations of same gel. See Fig. for complementary reactions with Grp94 N and Grp94 MC . See Fig. for uncropped gel images. ( B ) MS spectrum of crosslinked peptides containing the Grp94 K547 -OLF K468 linkage (top). HCD fragmentation spectrum and fragment mass match accuracy (1 of 9 PSMs observed) with y- and b-ion assignments (inset) consistent with the crosslinked peptides shown (bottom). Data correspond to the sample gel shown in Fig. ; refer to Table for additional details. ( C ) Map of the interaction interfaces between Grp94 and OLF identified by mass spectrometry. Protein domains demarcated by first amino acid residue in the domain (top, Grp94; bottom, OLF). Colors match those of the models in Fig. .

Article Snippet: Grp94 MC protein domain construct gene (residues 336–765, Canis lupus familiaris origin, 98.4% identity to human sequence for Grp94 MC , 97.9% identity overall based on Clustal Omega alignment ) was synthesized and codon-optimized for E . coli expression by ATUM and cloned into a pMAL-c5X vector (New England Biolabs) so that it contained a TEV-cleavable N-terminal maltose binding protein (MBP), and a Factor Xa-cleavable C-terminus 6xHis tag.

Techniques: Fluorescence, SDS Page, Mass Spectrometry, Residue

Journal: Scientific Reports

Article Title: Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation

doi: 10.1038/s41598-019-48751-8

Figure Lengend Snippet: Model of Grp94 protein-protein interaction sites with OLF. ( A ) Top: structural model of Grp94 highlighting both the aberrant (K72) and stabilizing (K547) interaction sites detected by crosslinking/MS (see Table ). Bottom: electrostatic surface potential view of boxed MC region; Grp94 MC model PDB-ID: 2O1T, which contains 18 additional, largely acidic C-terminal residues compared to 5ULS shown above (also see Fig. ). The proposed OLF recognition surface is identified by dotted circle. ( B ) Top: top-down perspective of OLF β-propeller, with unstructured N-terminal aggregation-associated Grp94 recognition site marked as Nterm*. Middle: side-on view of OLF with stabilizing interaction residue (K468) featured. Bottom: electrostatic surface potential of OLF in same pose as middle panel, indicating a positively-charged surface. The surface potential is colored negative (red, −5 kT/e − ) to positive (blue, 5 kT/e − ). The remaining color scheme and models shown are the same as in (Fig. ) unless otherwise specified.

Article Snippet: Grp94 MC protein domain construct gene (residues 336–765, Canis lupus familiaris origin, 98.4% identity to human sequence for Grp94 MC , 97.9% identity overall based on Clustal Omega alignment ) was synthesized and codon-optimized for E . coli expression by ATUM and cloned into a pMAL-c5X vector (New England Biolabs) so that it contained a TEV-cleavable N-terminal maltose binding protein (MBP), and a Factor Xa-cleavable C-terminus 6xHis tag.

Techniques: Residue

Journal: Scientific Reports

Article Title: Different Grp94 components interact transiently with the myocilin olfactomedin domain in vitro to enhance or retard its amyloid aggregation

doi: 10.1038/s41598-019-48751-8

Figure Lengend Snippet: The Pre-N region and conformational state of Grp94 N are critical for the aberrant interaction with myocilin OLF. ( A ) Truncation of the N domain of Grp94 eliminates its capacity to enhance the rate of OLF aggregation; residues 22-57 appear crucial to the interaction. Results represent the average of 12 replicates (for both OLF + Grp94 58-804 and OLF + Grp94 73-804 ) from 2 biological replicates. The ^ symbol represents data previously published ; ***(p < 0.0001) represents statistically significant differences relative to OLF + Grp94 FL . ( B ) Despite diminished effects on OLF aggregation, the majority of the Grp94 truncation variant proteins still co-aggregate with OLF. S = supernatant, W = wash, P = pellet/aggregate. ( C ) Treatment of Grp94 N with Grp94-specific inhibitor 4-Br-BnIm mitigates its interaction with OLF. The traces for Grp94 N (for comparison) are the same as in Fig. ; results for OLF + Grp94 N + 4-Br-BnIm represent the average of 9 replicates from 2 biological replicates. ***(p < 0.0001) represents statistically significant differences relative to OLF + Grp94 N . D) Grp94 N is partially rescued from its co-aggregation fate with OLF in the presence of inhibitor 4-Br-BnIm. Abbreviations given in ( B ).

Article Snippet: Grp94 MC protein domain construct gene (residues 336–765, Canis lupus familiaris origin, 98.4% identity to human sequence for Grp94 MC , 97.9% identity overall based on Clustal Omega alignment ) was synthesized and codon-optimized for E . coli expression by ATUM and cloned into a pMAL-c5X vector (New England Biolabs) so that it contained a TEV-cleavable N-terminal maltose binding protein (MBP), and a Factor Xa-cleavable C-terminus 6xHis tag.

Techniques: Variant Assay, Comparison