13 research outputs found
The Crystal Structure of Netrin-1 in Complex with DCC Reveals the Bifunctionality of Netrin-1 As a Guidance Cue
No full textSummaryNetrin-1 is a guidance cue that can trigger either attraction or repulsion effects on migrating axons of neurons, depending on the repertoire of receptors available on the growth cone. How a single chemotropic molecule can act in such contradictory ways has long been a puzzle at the molecular level. Here we present the crystal structure of netrin-1 in complex with the Deleted in Colorectal Cancer (DCC) receptor. We show that one netrin-1 molecule can simultaneously bind to two DCC molecules through a DCC-specific site and through a unique generic receptor binding site, where sulfate ions staple together positively charged patches on both DCC and netrin-1. Furthermore, we demonstrate that UNC5A can replace DCC on the generic receptor binding site to switch the response from attraction to repulsion. We propose that the modularity of binding allows for the association of other netrin receptors at the generic binding site, eliciting alternative turning responses
Structural analysis of the C-terminal region (modules 18-20) of complement regulator factor H (FH)
Get PDFFactor H (FH) is a soluble regulator of the human complement system affording protection to host tissues. It selectively inhibits amplification of C3b, the activation-specific fragment of the abundant complement component C3, in fluid phase and on self-surfaces and accelerates the decay of the alternative pathway C3 convertase, C3bBb. We have determined the crystal structure of the three carboxyl-terminal complement control protein (CCP) modules of FH (FH18-20) that bind to C3b, and which additionally recognize polyanionic markers specific to self-surfaces. These CCPs harbour nearly 30 disease-linked missense mutations. We have also deployed small-angle X-ray scattering (SAXS) to investigate FH18-20 flexibility in solution using FH18-20 and FH19-20 constructs. In the crystal lattice FH18-20 adopts a "J"-shape: A ~122-degree tilt between the structurally highly similar modules 18 and 19 precedes an extended, linear arrangement of modules 19 and 20 as observed in previously determined structures of these two modules alone. However, under solution conditions FH18-20 adopts multiple conformations mediated by flexibility between CCPs 18 and 19. We also pinpoint the locations of disease-associated missense mutations on the module 18 surface and discuss our data in the context of the C3b:FH interaction
Summary of FH19–20 SAXS data.
No full text<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<sub>g</sub></i> distribution from the ensemble analysis using EOM (pool in grey, selected ensemble in red).</p
Crystal structure of FH18–20 (PDB ID: 3SW0).
No full text<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
Crystallographic data collection and refinement statistics.
No full text<p>Values in parentheses are for the highest resolution shell. 5% of reflections were used as a test set for the calculation of R<sub>free</sub>.</p
FH18–20 SAXS analysis.
No full text<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<sub>g</sub></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<sub>g</sub></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
Overall SAXS parameters.
No full text<p><i>R<sub>g</sub><sup>Guinier</sup></i> and <i>R<sub>g</sub><sup>GNOM</sup></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<sub>max</sub></i> is the maximum particle dimension; <i>I(0)</i> is the forward scattering intensity; <i>MW<sup>(SAXS)</sup></i> is the molecular weight determined by SAXS; Vol<i><sup>SAXS</sup></i> is the hydrated particle volume of solutes determined from the SAXS patterns; and Vol<i><sup>DAM</sup></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
Location of disease-associated missense mutations within CCP 18.
No full text<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.
No full text<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
Structural basis for engagement by complement factor H of C3b on a self surface.
Get PDFComplement factor H (FH) attenuates C3b molecules tethered by their thioester domains to self surfaces and thereby protects host tissues. Factor H is a cofactor for initial C3b proteolysis that ultimately yields a surface-attached fragment (C3d) corresponding to the thioester domain. We used NMR and X-ray crystallography to study the C3d-FH19-20 complex in atomic detail and identify glycosaminoglycan-binding residues in factor H module 20 of the C3d-FH19-20 complex. Mutagenesis justified the merging of the C3d-FH19-20 structure with an existing C3b-FH1-4 crystal structure. We concatenated the merged structure with the available FH6-8 crystal structure and new SAXS-derived FH1-4, FH8-15 and FH15-19 envelopes. The combined data are consistent with a bent-back factor H molecule that binds through its termini to two sites on one C3b molecule and simultaneously to adjacent polyanionic host-surface markers
