c3b domain  (Hycult Biotech)

Journal: Frontiers in Immunology

Article Title: Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

doi: 10.3389/fimmu.2019.02097

Figure Lengend Snippet: Surface Plasmon Resonance (SPR) analysis showing interaction of properdin with C3b and the C3b/C3 CTC domain. SPR sensorgrams (left) and equilibrium binding plots (right). (A) Interaction of P N12/456 (concentration range: 4.9 × 10 −3 μM to 40 μM) with a C3b coated chip at physiological ionic strength (150 mM NaCl). (B) interaction of P N12/456 (concentration range: 1.2 × 10 −3 μM to 10 μM) with a C3b coated chip at low ionic strength (50 mM NaCl). (C) Interaction of properdin (concentration range: 1.2 × 10 −4 μM to 1 μM) with a C3b coated chip. (D) Binding of the C3/C3b CTC domain (concentration range: 2.4 × 10 −2 μM to 200 μM) to a P N1 ′/456 coated chip. The data point at 200 μM C3/C3b CTC was considered as an outlier and was not used to determine the K D . Where indicated Salp20 was used to regenerate the surface.

Article Snippet: P N1/456 and the C3/C3b-CTC domain were dialyzed overnight at 4°C using a 3.5 kDa cutoff Slide-A-Lyzer Mini Dialysis Unit (Thermo Scientific) against 10 mM HEPES, 50 mM NaCl, pH 7.4.

Techniques: SPR Assay, Binding Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

doi: 10.3389/fimmu.2019.02097

Figure Lengend Snippet: SPR analysis showing interaction of properdin with C3 pro-convertase. SPR sensorgrams (left) and equilibrium binding plots (right). C3bB dgf‡ and C3bBb dgf‡ were generated on the chip by injecting FB dgf‡ or FB dgf‡ and subsequently FD on a C3b coated chip. (A) Interaction of P N12/456 (concentration range: 2.4 × 10 −4 μM to 2 μM) with C3bB dgf‡ . (B) Interaction of P N12/456 (concentration range: 1.2 × 10 −4 μM to 1 μM) with C3bBb dgf‡ . (C) Interaction of properdin (concentration range: 6.1 × 10 −5 μM to 0.5 μM) with C3bB dgf‡ . (D) Interaction of properdin (concentration range: 6.1 × 10 −5 μM to 0.5 μM) with C3bBb dgf‡ . Where indicated Salp20, FD, and DAF were used to regenerate the surface.

Article Snippet: P N1/456 and the C3/C3b-CTC domain were dialyzed overnight at 4°C using a 3.5 kDa cutoff Slide-A-Lyzer Mini Dialysis Unit (Thermo Scientific) against 10 mM HEPES, 50 mM NaCl, pH 7.4.

Techniques: Binding Assay, Generated, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

doi: 10.3389/fimmu.2019.02097

Figure Lengend Snippet: Overview of properdin structures. (A) From left to right: P N1/456 , P N12/456 , and P N1/456 -CTC. Structures are depicted in cartoon representation with a semi-transparent molecular surface (top row) and as ADP cartoon putty (bottom row). ADP colors for all three structures are on the same scale of 10–140 Å 2 . (B) Cartoon representation of individual properdin domains; the TSR Trp-ladder residues, disulphides, and glycans are depicted as sticks. A schematic representation of the general TSR domain topology is included, showing the three strands and the position of the WxxWxxW and RxRxR motifs; the three disulphides are represented by dashed lines. TSR domains are shown with the Trp-ladder in approximately the same orientation. TSR1 and TSR2 were taken from FP N12/456 , TSR6 from FP N1/456 -CTC and STB, TSR4 & TSR5 from FP N1/456 . Unless stated otherwise, domains are colored as follows: STB (purple), TSR1 (blue), TSR2 (coral), TSR4 (yellow), TSR5 (green), TSR6 (red) from properdin, and the C3/C3b CTC domain (gray).

Article Snippet: P N1/456 and the C3/C3b-CTC domain were dialyzed overnight at 4°C using a 3.5 kDa cutoff Slide-A-Lyzer Mini Dialysis Unit (Thermo Scientific) against 10 mM HEPES, 50 mM NaCl, pH 7.4.

Techniques:

Journal: Frontiers in Immunology

Article Title: Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

doi: 10.3389/fimmu.2019.02097

Figure Lengend Snippet: Properdin-convertase interactions. (A) Surface representation of P N1/456 -CTC. Domains colored as follows: STB (purple), TSR1 (blue), TSR4 (yellow), TSR5 (green), TSR6 (red) from properdin, the C3/C3b CTC domain (gray), and Bb (brown). (B) Detailed view of the interaction between TSR5 and the C3/C3b CTC C-terminal α-helix. (C) Side view of P N1/456 -CTC, 90° rotated compared to (B) showing details of the interaction between the TSR5 and TSR6 stirrup loops and C3/C3b-CTC. In (B,C) proteins are shown in cartoon representation with side chains of key residues that are involved in the interaction shown in sticks. H-bonds are indicated as dashed lines. (D) Detail of the properdin-C3bBb-SCIN complex showing electron density at 1-rmsd contour level. (E) Close-up of the properdin-C3b-Bb interface showing the two properdin stirrup-loops that are sandwiched between C3b and Bb. Putative interaction in the properdin-C3bBb interface are shown as sticks. (F) TSR4 from all five properdin structures that are described in this paper: P N1/456 (yellow), P N12/456 (red), P N1/456 -CTC (green), and two copies of properdin in the properdin-C3bBb-SCIN complex (purple and pink) with models superposed using the distal part of TSR4 (residues 267–278 and 304–312).

Article Snippet: P N1/456 and the C3/C3b-CTC domain were dialyzed overnight at 4°C using a 3.5 kDa cutoff Slide-A-Lyzer Mini Dialysis Unit (Thermo Scientific) against 10 mM HEPES, 50 mM NaCl, pH 7.4.

Techniques:

Journal: Frontiers in Immunology

Article Title: Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

doi: 10.3389/fimmu.2019.02097

Figure Lengend Snippet: Models of properdin oligomers binding to surface bound C3 convertases. (A) Structures of P N1/456 (red), P N12/456 (yellow), P N1/456 -CTC (green), and the copy from Pc-C3bBb-SCIN lacking density for TSR3 (pink) superimposed on TSR5 of the other copy of Pc-C3bBb-SCIN (purple). (B) Ribbon representation of properdin oligomers binding to C3 convertases viewed from the front (left) and top (right). C3b and Bb are colored gray and wheat, respectively, Gln1013 from the C3b thioester is shown as red spheres. Each protomer in a properdin oligomers is colored differently. Top: Properdin dimer binding to two C3 convertases (for this model we used P N12/456 with TSR3 positioned relative to TSR2 as it is in the copy of Pc-C3bBb-SCIN that contains TSR3) Middle: Properdin trimer binding to 3 C3 convertases (for this model the properdin copy from Pc-C3bBb-SCIN that contains TSR3 was used). Bottom: Properdin tetramer binding to four C3 convertases (this model was generated with TSR2 as in the middle panel but using TSR4 from P N1/456 ).

Article Snippet: P N1/456 and the C3/C3b-CTC domain were dialyzed overnight at 4°C using a 3.5 kDa cutoff Slide-A-Lyzer Mini Dialysis Unit (Thermo Scientific) against 10 mM HEPES, 50 mM NaCl, pH 7.4.

Techniques: Binding Assay, Generated

Journal: Frontiers in Immunology

Article Title: Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

doi: 10.3389/fimmu.2019.02097

Figure Lengend Snippet: Properdin binding to C3b is incompatible with FI binding. Superposition of P N1/456 -CTC and C3b-FH-FI (PDB ID: 5O32). Models were superposed on the C3b-CTC domains (rmsd 0.7 Å). Left: overview of the structures with FH (Pink), FI (light blue), and properdin (multicolored model, with TSR5 in green and TSR6 in red) in ribbon presentation with semi-transparent molecular surface and C3b (gray) shown in ribbon. Right: close up showing FI occupies the same space as the properdin TSR6 (red) stirrup loop.

Article Snippet: P N1/456 and the C3/C3b-CTC domain were dialyzed overnight at 4°C using a 3.5 kDa cutoff Slide-A-Lyzer Mini Dialysis Unit (Thermo Scientific) against 10 mM HEPES, 50 mM NaCl, pH 7.4.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

doi: 10.3389/fimmu.2019.02097

Figure Lengend Snippet: Surface Plasmon Resonance (SPR) analysis showing interaction of properdin with C3b and the C3b/C3 CTC domain. SPR sensorgrams (left) and equilibrium binding plots (right). (A) Interaction of P N12/456 (concentration range: 4.9 × 10 −3 μM to 40 μM) with a C3b coated chip at physiological ionic strength (150 mM NaCl). (B) interaction of P N12/456 (concentration range: 1.2 × 10 −3 μM to 10 μM) with a C3b coated chip at low ionic strength (50 mM NaCl). (C) Interaction of properdin (concentration range: 1.2 × 10 −4 μM to 1 μM) with a C3b coated chip. (D) Binding of the C3/C3b CTC domain (concentration range: 2.4 × 10 −2 μM to 200 μM) to a P N1 ′/456 coated chip. The data point at 200 μM C3/C3b CTC was considered as an outlier and was not used to determine the K D . Where indicated Salp20 was used to regenerate the surface.

Article Snippet: The C3/C3b-CTC domain was purified on a Superdex 200 16/600 (GE Healthcare) in 20 mM HEPES pH 7.4, 150 mM NaCl.

Techniques: SPR Assay, Binding Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

doi: 10.3389/fimmu.2019.02097

Figure Lengend Snippet: SPR analysis showing interaction of properdin with C3 pro-convertase. SPR sensorgrams (left) and equilibrium binding plots (right). C3bB dgf‡ and C3bBb dgf‡ were generated on the chip by injecting FB dgf‡ or FB dgf‡ and subsequently FD on a C3b coated chip. (A) Interaction of P N12/456 (concentration range: 2.4 × 10 −4 μM to 2 μM) with C3bB dgf‡ . (B) Interaction of P N12/456 (concentration range: 1.2 × 10 −4 μM to 1 μM) with C3bBb dgf‡ . (C) Interaction of properdin (concentration range: 6.1 × 10 −5 μM to 0.5 μM) with C3bB dgf‡ . (D) Interaction of properdin (concentration range: 6.1 × 10 −5 μM to 0.5 μM) with C3bBb dgf‡ . Where indicated Salp20, FD, and DAF were used to regenerate the surface.

Article Snippet: The C3/C3b-CTC domain was purified on a Superdex 200 16/600 (GE Healthcare) in 20 mM HEPES pH 7.4, 150 mM NaCl.

Techniques: Binding Assay, Generated, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

doi: 10.3389/fimmu.2019.02097

Figure Lengend Snippet: Overview of properdin structures. (A) From left to right: P N1/456 , P N12/456 , and P N1/456 -CTC. Structures are depicted in cartoon representation with a semi-transparent molecular surface (top row) and as ADP cartoon putty (bottom row). ADP colors for all three structures are on the same scale of 10–140 Å 2 . (B) Cartoon representation of individual properdin domains; the TSR Trp-ladder residues, disulphides, and glycans are depicted as sticks. A schematic representation of the general TSR domain topology is included, showing the three strands and the position of the WxxWxxW and RxRxR motifs; the three disulphides are represented by dashed lines. TSR domains are shown with the Trp-ladder in approximately the same orientation. TSR1 and TSR2 were taken from FP N12/456 , TSR6 from FP N1/456 -CTC and STB, TSR4 & TSR5 from FP N1/456 . Unless stated otherwise, domains are colored as follows: STB (purple), TSR1 (blue), TSR2 (coral), TSR4 (yellow), TSR5 (green), TSR6 (red) from properdin, and the C3/C3b CTC domain (gray).

Article Snippet: The C3/C3b-CTC domain was purified on a Superdex 200 16/600 (GE Healthcare) in 20 mM HEPES pH 7.4, 150 mM NaCl.

Techniques:

Journal: Frontiers in Immunology

Article Title: Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

doi: 10.3389/fimmu.2019.02097

Figure Lengend Snippet: Properdin-convertase interactions. (A) Surface representation of P N1/456 -CTC. Domains colored as follows: STB (purple), TSR1 (blue), TSR4 (yellow), TSR5 (green), TSR6 (red) from properdin, the C3/C3b CTC domain (gray), and Bb (brown). (B) Detailed view of the interaction between TSR5 and the C3/C3b CTC C-terminal α-helix. (C) Side view of P N1/456 -CTC, 90° rotated compared to (B) showing details of the interaction between the TSR5 and TSR6 stirrup loops and C3/C3b-CTC. In (B,C) proteins are shown in cartoon representation with side chains of key residues that are involved in the interaction shown in sticks. H-bonds are indicated as dashed lines. (D) Detail of the properdin-C3bBb-SCIN complex showing electron density at 1-rmsd contour level. (E) Close-up of the properdin-C3b-Bb interface showing the two properdin stirrup-loops that are sandwiched between C3b and Bb. Putative interaction in the properdin-C3bBb interface are shown as sticks. (F) TSR4 from all five properdin structures that are described in this paper: P N1/456 (yellow), P N12/456 (red), P N1/456 -CTC (green), and two copies of properdin in the properdin-C3bBb-SCIN complex (purple and pink) with models superposed using the distal part of TSR4 (residues 267–278 and 304–312).

Article Snippet: The C3/C3b-CTC domain was purified on a Superdex 200 16/600 (GE Healthcare) in 20 mM HEPES pH 7.4, 150 mM NaCl.

Techniques:

Journal: Frontiers in Immunology

Article Title: Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

doi: 10.3389/fimmu.2019.02097

Figure Lengend Snippet: Models of properdin oligomers binding to surface bound C3 convertases. (A) Structures of P N1/456 (red), P N12/456 (yellow), P N1/456 -CTC (green), and the copy from Pc-C3bBb-SCIN lacking density for TSR3 (pink) superimposed on TSR5 of the other copy of Pc-C3bBb-SCIN (purple). (B) Ribbon representation of properdin oligomers binding to C3 convertases viewed from the front (left) and top (right). C3b and Bb are colored gray and wheat, respectively, Gln1013 from the C3b thioester is shown as red spheres. Each protomer in a properdin oligomers is colored differently. Top: Properdin dimer binding to two C3 convertases (for this model we used P N12/456 with TSR3 positioned relative to TSR2 as it is in the copy of Pc-C3bBb-SCIN that contains TSR3) Middle: Properdin trimer binding to 3 C3 convertases (for this model the properdin copy from Pc-C3bBb-SCIN that contains TSR3 was used). Bottom: Properdin tetramer binding to four C3 convertases (this model was generated with TSR2 as in the middle panel but using TSR4 from P N1/456 ).

Article Snippet: The C3/C3b-CTC domain was purified on a Superdex 200 16/600 (GE Healthcare) in 20 mM HEPES pH 7.4, 150 mM NaCl.

Techniques: Binding Assay, Generated

Journal: Frontiers in Immunology

Article Title: Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b

doi: 10.3389/fimmu.2019.02097

Figure Lengend Snippet: Properdin binding to C3b is incompatible with FI binding. Superposition of P N1/456 -CTC and C3b-FH-FI (PDB ID: 5O32). Models were superposed on the C3b-CTC domains (rmsd 0.7 Å). Left: overview of the structures with FH (Pink), FI (light blue), and properdin (multicolored model, with TSR5 in green and TSR6 in red) in ribbon presentation with semi-transparent molecular surface and C3b (gray) shown in ribbon. Right: close up showing FI occupies the same space as the properdin TSR6 (red) stirrup loop.

Article Snippet: The C3/C3b-CTC domain was purified on a Superdex 200 16/600 (GE Healthcare) in 20 mM HEPES pH 7.4, 150 mM NaCl.

Techniques: Binding Assay