Location of disease-associated missense mutations within CCP 18.

<p>A, Shown are the alpha-carbons (red spheres) of residues for which missense mutations associated with aHUS or basal laminar drusen have been reported. Residue numbers are: 1050 (basal laminar drusen variant N1050Y) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Boon1" target="_blank">[31]</a>; 1060 (aHUS-associated variant V1060A) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Guigonis1" target="_blank">[36]</a>; 1076 (aHUS-associated variant Q1076E) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Perkins1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Richards1" target="_blank">[34]</a>; and 1078 (basal laminar drusen-associated variant R1078S) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Boon1" target="_blank">[31]</a>. Also indicated in magenta is the alpha-carbon of residue 1095, the Asn of the sole N-glycosylation consensus sequence located within FH18–20. B, Electrostatic surface representation of FH18–20. Positively and negatively charged areas are indicated in blue and red, respectively. Also shown as a red mesh is a negative isosurface map contoured at −2 kT/e. This figure was generated using the APBS plug-in for PyMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Baker1" target="_blank">[64]</a>.</p

The crystal structure of FH18–20 modeled onto the C3b:FH1–4 complex.

<p>A, Superposition of FH18–20 structure (PDB ID: 3SW0) on the previously determined wild-type FH19–20:C3d complex (PDB ID: 3OXU <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Morgan1" target="_blank">[18]</a>) and the FH1–4:C3b complex (PDB ID: 2WII <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Wu1" target="_blank">[15]</a>). Surface representations are shown of FH1–4 (slate) and FH18–20 (cyan). In the cartoon representation of C3b, constituent domains are color-coded with the TED indicated in green. FH19–20 and C3d were employed for alignment purposes only, and are not shown. Also indicated is Gln1013, the site of covalent linkage of C3b to target surfaces. B, As for (A) except the model of the FH1–4:C3b:FH18–20 complex has been rotated about the <i>y</i>-axis by 35° demonstrating the path of CCP 18 with respect to the FH1–4:C3b complex. C, Schematic of a FH1–4:C3b:FH18–20 complex demonstrating the inferred flexibility, in solution, of the linker connecting CCPs 18 and 19.</p

Summary of FH19–20 SAXS data.

<p>A, Superposition of the SAXS-derived shape envelope of recombinant FH19–20 (shown in yellow) on the crystal structure of FH19–20 (PDB ID: 3OXU <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Morgan1" target="_blank">[18]</a>). Shape envelopes were determined using the <i>ab initio</i> bead-modelling program DAMMIF <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Franke1" target="_blank">[48]</a> and superposition of the FH19–20 envelope on the corresponding crystal structure was carried out utilizing the program, SUPCOMB13 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Kozin1" target="_blank">[50]</a>. B, Fit of the X-ray crystal structure of FH19–20 (solid black line) to the SAXS data extrapolated to infinite dilution (black open circles). The fit of the selected ensemble of conformations from EOM is also shown (solid red line). C, The <i>R_g</i> distribution from the ensemble analysis using EOM (pool in grey, selected ensemble in red).</p

Crystallographic data collection and refinement statistics.

<p>Values in parentheses are for the highest resolution shell. 5% of reflections were used as a test set for the calculation of R_free.</p

Crystal structure of FH18–20 (PDB ID: 3SW0).

<p>A, A Cartoon representation of the three CCP modules is indicated: CCP 18 (residues 1048–1102), CCP 19 (residues 1109–1163), and CCP 20 (residues 1167–1228). Highlighted on the FH18–20 structure are the C3b-binding (green) and polyanion-binding (blue) regions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Kajander1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Morgan1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Herbert1" target="_blank">[21]</a>. Residues contributing to the inter-domain packing between CCPs 18 and 19 are shown. B, Close-up of the kink that occurs between modules 18 and 19. The orientation of the FH18–20 molecule is the same as shown in ‘A’. Electron density (2<i>F</i>o−<i>F</i>c map shown in grey, and contoured at 1.5σ) for residues contributing to the inter-modular packing is shown. Dashed lines represent hydrogen-bonds between amino acid residues or between amino acid residues and water molecules. C, as for ‘B’ except the molecule is rotated about the <i>y</i>-axis by 180°.</p

Overall SAXS parameters.

<p><i>R_g^Guinier</i> and <i>R_g^GNOM</i> are the experimentally determined radius of gyration as calculated by Guinier analysis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Guinier1" target="_blank">[62]</a> and by indirect Fourier transform using the program GNOM, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Svergun2" target="_blank">[63]</a>; <i>D_max</i> is the maximum particle dimension; <i>I(0)</i> is the forward scattering intensity; <i>MW^(SAXS)</i> is the molecular weight determined by SAXS; Vol<i>^SAXS</i> is the hydrated particle volume of solutes determined from the SAXS patterns; and Vol<i>^DAM</i> is the excluded volume of solutes determined using the <i>ab initio</i> modeling program DAMMIF <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032187#pone.0032187-Franke1" target="_blank">[48]</a>. Data merged and extrapolated to infinite dilution are referred to in the table as “mer”.</p

FH18–20 SAXS analysis.

<p>A, Superposition of the SAXS-derived shape envelope of recombinant FH18–20 (yellow) on the crystal structure of FH18–20 (indicated in cyan), and also on a CORAL-derived rigid body model of FH18–20 (shown in red) where the orientation of CCPs 19–20 are fixed, and the position of CCP 18 refined against the SAXS data. Flexible linker residues (alpha-carbon atoms) are shown as red spheres. B, Fit of the FH18–20 crystal structure (black line) to the SAXS data using the program CRYSOL, and fit of the selected ensemble of FH18–20 models from the EOM analysis (red line) to the SAXS data. C, <i>R_g</i> distribution from the EOM analysis of FH18–20 with both FH18–19 and FH19–20 linker regions defined as flexible (pool in grey, selected ensemble in red). D, Fits of the selected ensembles from the EOM analysis of FH18–20 to the SAXS data using flexible FH18–19 (blue line) or FH19–20 (green line) linker regions. E, <i>R_g</i> distribution from the EOM analysis for FH18–20 with the FH18–19 linker region defined as flexible (pool in grey, selected ensemble in blue).</p

Backbone dynamics and chemical shift-based secondary structure of Hsp12.

<p><i>T</i>₁, <i>T</i>₂ and <i>T</i>₁/<i>T</i>₂ relaxation values are shown for Hsp12 in the presence (A,C,E) and absence (B,D,F) of 100 mM SDS at 318 K. <i>T</i>₁ and <i>T</i>₂ relaxation times for micelle-bound (A,C) Hsp12 show significant variation; contrasting with the similar relaxation values observed for free Hsp12 (B,D). Micelle-bound Hsp12 (E) shows grouped variations in the <i>T</i>₁/<i>T</i>₂ values ranging from approximately 1.5 to 14, indicating a wide range of mobility and a clear differentiation of secondary structure elements; whereas the free form (F) shows consistent values of around 2, indicating a completely unstructured protein. (G) The assigned chemical shifts at 318 K in 100 mM SDS expressed as deviation from random coil are shown aligned with the primary sequence and the positions of the α-helices.</p

Hsp12 is unstructured in solution, but folds in the presence of SDS.

<p>(A) ¹H-¹⁵N HSQC spectrum of Hsp12 in aqueous solution at 298 K. The spectrum shows only sharp peaks with random coil shifts indicating the absence of any structured regions. (B) ¹H-¹⁵N HSQC spectrum of Hsp12 at 303 K in the presence of increasing concentrations of SDS (0, 1, 2, 5, 8 mM Red -> Blue). SDS causes a considerable increase in the amount of chemical shift dispersion implying increased levels of folded material/regions. (C) Assigned ¹H-¹⁵N HSQC spectrum of Hsp12 at 318 K in the presence of 100 mM SDS.</p

Helical properties of micelle-bound Hsp12.

<p>(A) The four α-helices are represented as ribbons and colour coded from the N-terminus (blue) to the C-terminus (red) in a representative structure. (B,C) Analysis of charge distribution with hydrophobic residues labelled green and charged residues labelled red in both ribbon (B) and surface (C) representation, illustrating the amphipathic nature of Hsp12. Structures were generated using Chimera.</p