Figure 1. Principles of properdin architecture and the structure of FP1.

(A) Schematic representation of the FP1 monomer and FP2 as an example of an oligomer, where one subunit is colored gray, while the other is colored according to the domain structure as for FP1. (B) The 10 most populated nsEM 2D classes obtained with FP1 with the number of particles indicated. A magnified view of the 2D class marked by star is shown to the right. (C) As for (B), but for the FP1-hFPNb1 complex. Compared to FP1, an additional mass marks the location of hFPNb1 and hence TSR4 enabling assignment of the TB domain and the six thrombospondin repeats in the magnified view to the right. (D) Representative atomic model of FP1 E244K derived by rigid-body modeling against the SAXS data. (E) Comparison of SAXS experimental data (Pedersen et al., 2017) and the fitted scattering curve corresponding to the FP1 E244K model presented in (E). Insert to (E): p(r) function derived from the SAXS data. (F) Conformational ensemble of FP1 E244K sampled by a 1 μs MD simulation represented by 100 frames with 10 ns interval shown as transparent tubes. The starting model is displayed as a cartoon with the glycans and glycosylated residues in gray stick representation. Disulfide bridges are represented by yellow sticks. (G) Comparison of SAXS experimental data and the scattering curve obtained from MD ensemble after refinement using the Bayesian maximum entropy approach, the two curves fit with χ² = 1.4. The minor difference in the experimental data apparent at the highest q-values in (E) and (G) is due to subtraction of a constant by CORAL in (E).

Figure 1—figure supplement 1. Elution profiles of recombinant FP.

(A) Top, SEC analysis of initial pool of recombinant FP obtained by His₆–based affinity chromatography. Below is presented SEC profiles for the final purification step of recombinant FP1, FP2, FP3, and FP4 obtained from HEK293F cells used for SAXS data collection and negative-stain EM (grayed areas). (B) SEC profile of commercial plasma FP for comparison. (C–E) SEC analysis of FP1, FP2, and FP3 with or without hFPNb1 used for nsEM in Figures 1 and 2. (F–G) SEC analysis of the FP2/FP3 pool and a pool dominated by FP2 used the functional assays in Figure 5 and for the exchange experiments in Figure 4, respectively.

Figure 1—figure supplement 2. Molecular dynamics simulation of the FP1 E244K.

(A,B) The root mean square deviation of each domain and the whole protein with the trajectories aligned to the initial FP1 E244K model in two independent 1 µs MD simulations. Both simulations commonly revealed significant dynamics of TSR3. (C) The RMS fluctuations within the entire FP E244K with the trajectory aligned to the initial model illustrated by cartoon representations in which the thickness of the tube is scaled by the corresponding RMS fluctuation values per residue. (D–F) Close-up of the RMS fluctuations of TSR3, TSR2, and TSR4 with the trajectories aligned to the corresponding domain, indicating that these thrombospondin repeats have limited conformational flexibility in their overall structure. Importantly, the mutation of glutamate 244 to lysine in TSR3 does not cause breakdown of the central Trp-Arg stack.

Figure 1—video 1. Molecular dynamics simulation of FP E244K.

Download video file ^{(10.3MB, mp4)}

Domains and glycans are colored as in Figure 1F. All frames in a 1 µs simulation are displayed as thin lines in the background. Notice how TSR3 is able to rotate substantially around the two hinges connecting it to TSR2 and TSR4 facilitated by flexibility of the N-terminal regions in TSR3 and TSR4. The rest of the molecule exhibits only limited dynamics.