Biophysical Studies of the Induced Dimerization of Human VEGF Receptor 1 Binding Domain by Divalent Metals Competing with VEGF-A

Abstract

Angiogenesis is tightly regulated through the binding of vascular endothelial growth factors (VEGFs) to their receptors (VEGFRs). In this context, we showed that human VEGFR1 domain 2 crystallizes in the presence of Zn2+, Co2+ or Cu2+ as a dimer that forms via metal-ion interactions and interlocked hydrophobic surfaces. SAXS, NMR and size exclusion chromatography analyses confirm the formation of this dimer in solution in the presence of Co2+, Cd2+ or Cu2+. Since the metal-induced dimerization masks the VEGFs binding surface, we investigated the ability of metal ions to displace the VEGF-A binding to hVEGFR1: using a competition assay, we evidenced that the metals displaced the VEGF-A binding to hVEGFR1 extracellular domain binding at micromolar level.

Fig 1. Crystal structure of hVEGFR1d2 homodimers.

Zn²⁺-dimer (light blue and green) (4CKV), the Co²⁺-dimer (salmon and magenta) (4CL7) and the Cu²⁺-dimer (orange and purp(A) Interface of the Zn²⁺-induced dimer. The hVEGFR1d2 buried side chains from one monomer are represented as stick surrounded by a dot surface. The second monomer is represented by its surface conventionally colored according to the electrostatic potential. (B) The crystal le) (5ABD) were superimposed on one of the two molecules (domain2). The second molecule of the three dimers are represented on the right side: Zn²⁺-domain2-sym is rotated by 7° and translated by 1.1 Å in regard to Co²⁺-domain2-sym. The Zn²⁺-domain2-sym is rotated by 12° and translated by 2.4 Å in regard to Cu²⁺-domain2-sym. However, the residues in contact at the interface remain unchanged and their side chains are shown as sticks. (C) Metal coordination for Zn²⁺, Co²⁺ and Cu²⁺.

Fig 2. The hVEGFR1d2 dimerization interface overlapped the VEGF binding site.

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.

Fig 3. SAXS analysis of hVEGFR1d2 in the absence (A, B) and presence (C, D) of CoCl₂.

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

Fig 4. ¹H-¹⁵N-TROSY NMR spectra of the hVEGFR1d2 domain.

(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.

Fig 5. Peak volume evolution of the 1H-15N TROSY with increasing concentrations of CdCl₂.

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.

Fig 6. hVEGFR1-d2d3 structural modeling.

(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).

Fig 7. Size-exclusion chromatography reveals the Cu²⁺ induced hVEGFR1d2 dimerization.

(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).