The allosteric modulation of complement C5 by knob domain peptides
- UCB, United Kingdom
- Department of Biology and Biochemistry, University of Bath, United Kingdom
- European Molecular Biology Laboratory, Hamburg Unit, Germany
- Department of Statistics, University of Oxford, United Kingdom
- Department of Chemistry and Biomedicine, Linnaeus University, Sweden
- Department of Immunology, Oslo University Hospital, University of Oslo, Norway
- Research Laboratory, Bodø Hospital, K.G. Jebsen TREC, University of Tromsø, Norway
- Centre of Molecular Inflammation Research, Norwegian University of Science and Technology, Norway
- Centre for Therapeutic Innovation, University of Bath, United Kingdom
Figures
Figure 1
Functional modulation of C5 via knob domain peptides.
(A) shows an abridged schematic for classical pathway (CP) activation. Following activation of C1q via antibody Fc, C4 and C2 are cleaved and form C4bC2a (the CP C3 convertase) which cleaves C3 into …
Figure 2
Surface plasmon resonance peptide cross blocking.
(A), (B), and (C) highlight negative cooperativity between the K8, K92, and K57 peptides, respectively. Neither K57 or K92 can bind to the C5-K8 complex but K8 can bind, albeit at a lower level, to …
Figure 3 with 4 supplements
Crystal structures of C5-knob domain complexes.
(A) and (B) show the crystal structures of C5 in complex with the K8 and K92 knob domain peptides, respectively. The binding site for the K8 peptide (A, shown in red) is located on a previously …
Figure 3—figure supplement 1
Structural analysis of the C5-K8 complex.
(A) Structural topology of the K8 peptide. Topology diagram highlighting the secondary structural elements present in the K8 peptide. This image was generated using PDBsum (EMBL-EBI). (B) K8-C5 …
Figure 3—figure supplement 2
Simulated annealing OMIT maps of the C5-knob domain peptide complexes.
The blue mesh shows mFo-DFc simulated annealing OMIT maps calculated in PHENIX and contoured at (A) 3.0 σ around the K8 peptide and (B) at 1.3 σ around the K92 peptide. In the OMIT calculation, the …
Figure 3—figure supplement 3
Structural analysis of the C5-K92 complex.
(A) Structural topology of the K92 peptide. Topology diagram of the K92 peptide highlighting the secondary structural elements present in the K92 peptide. This image was generated using PDBsum …
Figure 3—figure supplement 4
Paratope analysis of K8 and K92 peptides.
(A) Comparison of K8 and K92 paratope size with known antibody–antigen complexes. K8 and K92 paratope size are compared to non-redundant antibodies in the structural antibody database SAbDab (N = 924…
Figure 4 with 1 supplement
Comparison of the K8 and K92 binding sites with known C5 inhibitor complexes.
Structural alignment of the complexes of C5 with the K8 and K92 knob domain peptides with the known structures for OmCI and RaCI (Protein Data Bank [PDB] accession code 5HCC; Jore et al., 2016), …
Figure 4—figure supplement 1
Structural comparison of C5-K92 and C5-CVF complexes.
Structures of the C5-K92 and C5-CVF (Protein Data Bank accession code 3PRX; Laursen et al., 2011) complexes were superimposed via their MG5 domains, similar to those shown in Figure 4C. Top and …
Figure 5 with 1 supplement
Hydrodynamic properties and solution conformation of C5 and C5-knob domain complexes by small-angle X-ray scattering (SAXS).
Size exclusion chromatography multi-angle laser light scattering (SEC-MALLS) chromatograms (A) for apo C5 (black) and C5-K92 (orange) show a homogenous molecular weight increase across the C5-K92 …
Figure 5—figure supplement 1
SAXS analyses of the C5-knob domain peptide complexes.
(A) Size exclusion chromatography multi-angle laser light scattering (SEC-MALLS) and SEC small-angle X-ray scattering (SEC-SAXS) chromatograms for C5-knob domain complexes. SEC-MALLS chromatograms …
Figure 6 with 1 supplement
Impact of knob domain binding on the structural dynamics and conformation of C5.
Differential hydrogen-deuterium exchange (ΔHDX) plots for C5 in complex with knob domains (A) K8, (B) K57, and (C) K92 at 1 hr of deuterium exposure. Blue denotes peptides with decreased HDX …
Figure 6—figure supplement 1
HDX analyses of the C5-knob domain peptide complexes.
(A) Woods plot displaying the differential hydrogen-deuterium exchange (ΔHDX) for C5 in complex with knob domains. (A) K8, (B) K57, and (C) K92 at four deuterium exposure time points: 0.5 min, 2 …
Additional files
-
Supplementary file 1
Section 1. Functional analysis. Table 1.1. Classical pathway C5b deposition ELISA; Table 1.2. Alternative pathway C5b deposition ELISA; Table 1.3. Inhibition of classical pathway-mediated C5a release; Table 1.4. Inhibition of alternative pathway-mediated C5a release; Table 1.5. Inhibition of classical pathway-mediated C9 deposition; Table 1.6. Inhibition of alternative pathway-mediated C9 deposition; Table 1.7. Inhibition of classical pathway haemolysis; Table 1.8. Inhibition of alternative pathway haemolysis. Section 2. Structural analysis. Table 2.1. Data collection and refinement statistics (molecular replacement); Table 2.2. Hydrogen bond interactions between K8 and C5; Table 2.3. Ionic interactions between K8 and C5; Table 2.4. Disulphide mapping of the K92 peptide; Table 2.5. Hydrogen bond interactions between K92 and C5; Table 2.6. Validation of molecular interactions by peptide mutagenesis analysis; Table 2.7. Individual, total, and average hydrogen bond persistence in a binding pose metadynamics simulation of the K8-C5 complex; Table 2.8. Individual, total, and average hydrogen bond persistence in a binding pose metadynamics simulation of the K92-C5 complex. Section 3. Solution structure analysis. Table 3.1. SAXS Summary data; Table 3.2. ΔHDX summary data. Section 4. Additional functional analyses. Table 4.1. SPR single-cycle kinetics of knob domains binding to human C5b; Table 4.2. SPR single-cycle kinetics of knob domains binding to human C5b-6.
- https://cdn.elifesciences.org/articles/63586/elife-63586-supp1-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/63586/elife-63586-transrepform-v2.docx
