Size-exclusion chromatography reveals the Cu²⁺ induced hVEGFR1d2 dimerization.

<p>(A) hVEGFR1d2 eluted either in the presence of either 1 mM EDTA (Purple curve) or 1 mM CuSO₄ (Green curve). The chromatogram revealed a single peak in the presence of 1 mM EDTA (d), and two major peaks corresponding to the monomer (b) and the dimer (a) in the presence of Cu²⁺. A third peak (c) consisting in Cu²⁺ ions eluted near the total volume. (B) SDS page of loaded samples and eluted peaks. hVEGFR1-d2 + CuCl₂ (Green): Loaded sample (ls), Ve = 11.3 ml (a), Ve = 14.1 ml (b); hVEGFR1-d2 + EDTA (Purple): Loaded sample (ls), Ve = 17.3 ml (c), Ve = 14.0 ml (d).</p

hVEGFR1-d2d3 structural modeling.

<p>(A) hVEGFR1d2d3 model after MD simulation: two extreme conformers observed during the MD simulation are superimposed on domain 2 to illustrate the 40° hinge motion. (B) Top: crystal structure of the VEGFR2-d2d3/VEGF-E complex (3V6B). Bottom: Zn²⁺-dimerized hVEGFR1d2d3 model after MD simulation. The Zn²⁺ ion is shown as an orange sphere. Equivalent orientations of domain 3 are shown in identical colors (red or green).</p

Peak volume evolution of the 1H-15N TROSY with increasing concentrations of CdCl₂.

<p>The cadmium concentration was increased from 0 to 1.8 equivalents. The peak volumes are normalized against the highest peak volume in the TROSY experiment without cadmium. No bar indicates either the presence of proline or a residue (Asn₂₁₂) that could not be unambiguously identified on the spectrum. Extremely perturbed peaks following cadmium addition identify three principal regions potentially involved in dimerization: Tyr₁₃₉-Gly₁₅₁, Ala₁₉₇-Leu₂₀₄ and Leu₂₂₁-Thr₂₂₆.</p

displacement of VEGF-A binding to the VEGFR1-ECD by divalent cations in competition assay.

<p>IC₅₀s and % inhibition of assayed metal complexes.</p

¹H-¹⁵N-TROSY NMR spectra of the hVEGFR1d2 domain.

<p>(A) Overlay of the TROSY spectra of the hVEGFR1d2 at the concentration of 150 μM in the absence (red) and presence (black) of cadmium (1.8 eq). The full spectra are shown except for residues Phe₁₃₅ and Trp₁₈₆, with resonances at 5.6 ppm and 10.5 ppm, respectively. A general line broadening effect is observed following the addition of the divalent cation due to the dimerization of the hVEGFR1d2 and peaks of residues at the interface completely disappear due to the conformational exchange on an intermediate NMR timescale. (B) EDTA has been added (1.8 eq, 270 μM) to the precedent mixture. EDTA chelates cadmium ions, leading to the disruption of the dimer and the reemergence of the previously missing resonances characterizing the interface.</p

SAXS analysis of hVEGFR1d2 in the absence (A, B) and presence (C, D) of CoCl₂.

<p>(A) and (C): scattering intensities with the CRYSOL and DAMMIF fit, and pair-distribution function (P(r)); (B) and (D): corresponding SAXS <i>ab initio</i> DAMMIF shapes. The cobalt ion is shown as a black sphere on (D).</p

Crystal data collection and refinement statistics.

<p>Crystal data collection and refinement statistics.</p

SAXS data recording and analysis.

<p>SAXS data recording and analysis.</p

The hVEGFR1d2 dimerization interface overlapped the VEGF binding site.

<p>hVEGFR1d2 surface colored as a function of the buried surface area calculated by the program PISA, within several complexes: (A) homodimer hVEGFR1d2/hVEGFR1d2 (4CKV), (B) hVEGFR1d2/tVEGF-A (1FLT), (C) hVEGFR1d2/VEGF-B (2XAC), (D) hVEGFR1d2/PlGF (1RV6); (E) ribbon representation of the hVEGFR1d2 in the identical orientation. The homodimer interaction surface (A) mimics an important part of the hVEGFR1-ligand interaction surface (B, C, D), with a major contribution of Leu₂₂₁.</p