Journal: Nature Communications

Article Title: Insight into mode-of-action and structural determinants of the compstatin family of clinical complement inhibitors

doi: 10.1038/s41467-022-33003-7

Figure Lengend Snippet: a Cp40-C3b structure solved at 2.0-Å resolution showing a single molecule of Cp40 (stick representation) binding to the β-chain of C3b (green, cartoon representation; the α-chain is shown in cyan). b Cp40 binding site at the MG core of C3b highlighted in red. c Superimposition of the current Cp40-C3b structure (green/cyan) with the previous Cp01-C3c structure (grey). The dotted box shows an enlarged view of the compstatin binding region at the interface of the MG4 and MG5 domains of C3b/C3c. The two compstatin analogs are shown as sticks. d – f Structure of Cp40 in its target-bound conformation. d Cp40 is bound as cyclic 14-amino-acid peptide (green stick representation) engaging in intermolecular contacts with the MG4 (cyan) and MG5 (purple) domains of C3b. e Backbone trace (main chain as cartoon, side chain as sticks) revealing a twisted, O-shaped bound conformation of the disulfide-bridged peptide f Superimposition of Cp01 (grey; from C3c-Cp01 complex) and Cp40 (green; from C3b-Cp40 complex) showing an overall structural similarity with deviations primarily found in side chains not engaging in tight intermolecular interactions. CTC C-terminal complement domain, CUB complement C1r/C1s, Uegf Bmp1 domain, MG macroglobulin domain, TED thioester-containing domain.

Article Snippet: Human C3, C3b, FH, FB, and FD used in binding and functional studies were purchased from Complement Technology (Tyler, TX, USA); C3c for ITC experiments was obtained from Lee Biosolutions (Maryland Heights, MO, USA).

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Insight into mode-of-action and structural determinants of the compstatin family of clinical complement inhibitors

doi: 10.1038/s41467-022-33003-7

Figure Lengend Snippet: a Contact residues on the target protein engaging in H-bonds (yellow), lipophilic contacts (blue) or both (purple) are highlighted on the surface of C3b. Contacts only present in Cp01 but not Cp40 are show in pastel shades. The structures of Cp01 and Cp40 are shown in grey and green ball-and-stick representation, respectively; residues not engaging in target binding are shown as lines. c Intramolecular contact networks in Cp01 (left) and Cp40 (right) showing H-bonds in yellow and hydrophobic contacts in grey. Analog-specific H-bonds and hydrophobic contacts are marked yellow and cyan arrows, respectively. b , d Differences in binding free energies per residue between Cp01-C3c and Cp40-C3b based on three independent MD simulation runs with distinct initiation parameters. (Supplementary Tables – ). Closeup view of the interaction profiles of e D-Tyr1, f (1Me)Trp5, g Sar9, h mIle14, i Asp7, and j Arg12. C3b is shown in stick representation with grey semitransparent surface and Cp40 in green ball-and-stick representation. H-bonds and hydrophobic contacts are marked as yellow and brown dotted lines, respectively.

Article Snippet: Human C3, C3b, FH, FB, and FD used in binding and functional studies were purchased from Complement Technology (Tyler, TX, USA); C3c for ITC experiments was obtained from Lee Biosolutions (Maryland Heights, MO, USA).

Techniques: Binding Assay, Residue

Journal: Nature Communications

Article Title: Insight into mode-of-action and structural determinants of the compstatin family of clinical complement inhibitors

doi: 10.1038/s41467-022-33003-7

Figure Lengend Snippet: a Compstatin-based drugs may interact with circulating (i.e., C3, C3c) or surface-tethered targets (i.e., C3b, C3bBb, iC3b). Depending on their valency, avidity may occur. b ITC analysis of solution interactions between C3c and compstatin analogs Cp01, Cp05*, Cp05-PEG-Cp05, and Cp40 shows 1:1 binding for monovalent analogs ( N = 1) whereas the data of the bivalent Cp05-PEG-Cp05 fits a 2:1 ratio ( N = 0.5). The displayed curve is a representative of two independent experiments per compound, obtained by peak-to-peak integration of the processed thermogram, as a function of At/Mt (titrant-to-titrate molar ratio). The integrated signal (black) is fitted to a 1:1 binding model (red curve) for determination of affinity (K D ) and molecular ratio ( N ). Error bars represent the uncertainty associated to the integral calculation of each peak in the binding isotherm (as determined by AFFINImeter). Cp05* contains the linker necessary for the conjugation to PEG-40kDa; its binding profile is comparable to that of Cp05 (Supplementary Fig. ). c SPR analysis between immobilized C3b and mono- and bivalent Cp05 analogs reveal a strong enhancement of surface binding for Cp05-PEG-Cp05 over monovalent Cp05. d Isoaffinity kinetic plot for surface interactions of compstatin analogs with immobilized C3b, demonstrating that the development steps from Cp01 to Cp40 were enabled by improving dissociation rates with little impact on association rates. In contrast, the improved stability of the C3b complex with Cp05-PEG-Cp05 is accompanied by a marked drop in the association rate. PEG polyethylene glycol, RU resonance units.

Article Snippet: Human C3, C3b, FH, FB, and FD used in binding and functional studies were purchased from Complement Technology (Tyler, TX, USA); C3c for ITC experiments was obtained from Lee Biosolutions (Maryland Heights, MO, USA).

Techniques: Binding Assay, Conjugation Assay