Structural basis for selective vascular endothelial growth factor-A (VEGF-A) binding to neuropilin-1

Abstract

Neuropilin-1 (Nrp1) is an essential receptor for angiogenesis that binds to VEGF-A. Nrp1 binds directly to VEGF-A with high affinity, but the nature of their selective binding has remained unclear. Nrp1 was initially reported to bind to the exon 7-encoded region of VEGF-A and function as an isoform-specific receptor for VEGF-A(164/165). Recent data have implicated exon 8-encoded residues, which are found in all proangiogenic VEGF-A isoforms, in Nrp binding. We have determined the crystal structure of the exon 7/8-encoded VEGF-A heparin binding domain in complex with the Nrp1-b1 domain. This structure clearly demonstrates that residues from both exons 7 and 8 physically contribute to Nrp1 binding. Using an in vitro binding assay, we have determined the relative contributions of exon 7- and 8-encoded residues. We demonstrate that the exon 8-encoded C-terminal arginine is essential for the interaction of VEGF-A with Nrp1 and mediates high affinity Nrp binding. Exon 7-encoded electronegative residues make additional interactions with the L1 loop of Nrp1. Although otherwise conserved, the primary sequences of Nrp1 and Nrp2 differ significantly in this region. We further show that VEGF-A(164) binds 50-fold more strongly to Nrp1 than Nrp2. Direct repulsion between the electronegative exon 7-encoded residues of the heparin binding domain and the electronegative L1 loop found only in Nrp2 is found to significantly contribute to the observed selectivity. The results reveal the basis for the potent and selective binding of VEGF-A(164) to Nrp1.

Figures

FIGURE 1.

Crystal structure of VEGF-A HBD in complex with Nrp1.A, chain A (green) and chain B (blue) crystallized in an antiparallel fashion with the chain A VEGF-A HBD fully engaging the Nrp1-b1 domain of chain B, and that of chain B engaged by the symmetry related Nrp1-b1 domain of chain A. B, intermolecular complex enclosed in dashed box. A space-filling model revealing the specific interface with VEGF-A164 exons 7 and 8 (chain A) encoded residues with Nrp1 (chain B). C, stereo view of the 2Fo − Fc electron density map contoured at 1.0 σ of Nrp1 (gold) and exons 7 (blue) and 8 (green) of VEGF-A164. An intramolecular salt bridge between Asp-142 and Arg-163 of VEGF-A164 and an intermolecular salt bridge between Asp-320 of Nrp1 and Arg-164 of VEGF-A164 are observable. D, stereo view of the Fo − Fc omit electron density map for the HBD residues contoured at 3.0 σ.

FIGURE 2.

VEGF-A164 binds to Nrp1 with high affinity. VEGF-A164 (black line) binds Nrp1 with a Kd = 3.0 nm ± 0.2 nm. VEGF-A120 (gray dashed line) binds Nrp1 with a Kd = 22 nm ± 1 nm.

FIGURE 3.

Exon 8-encoded C-terminal arginine of VEGF-A mediates high affinity Nrp1 binding.A, Arg-164 forms specific contacts with the b1 binding pocket of Nrp1. The guanidinium moiety forms a salt bridge with Asp-320 carboxylate oxygens (dashed red lines, 3.08 Å and 3.32 Å). The C terminus forms hydrogen bonds with three Nrp1-b1 residues (dashed gray lines, 3.08 Å, 2.95 Å, and 3.13 Å to Ser-346, Thr-349, and Tyr-353, respectively). B, Tuftsin binds to the Nrp1-b1 domain (PDB code 2ORZ) utilizing the same C-terminal arginine binding mode. C, mutagenesis demonstrates a specific role for the side chain (R164A, black bar) and C terminus (R164R + AA, gray bar) in Nrp1 binding. Charge reversal (R164E, red bar) completely abolishes binding to Nrp1. Statistical comparison of mean wild-type and mutant binding demonstrates significant differences, p < 0.0002, between all constructs. AP-tagged wild-type and mutant VEGF were used at an activity of 25 μmol of pNPP hydrolyzed/min/μl. D, surface representation of VEGF-A bound to Nrp1 reveals the critical location of Thr-316 (red) and illustrates the mechanism by which mutation to arginine would occlude binding. E, occlusion of the Nrp1 binding pocket in the Thr-Arg Nrp1 mutant (T316R, red bar) completely abolishes binding to VEGF-A164. AP-tagged wild-type and mutant Nrp1 were used at an activity of 1 μmol of pNPP hydrolyzed/min per μl. Error bars, S.D.

FIGURE 4.

Glu-154 of VEGF-A164 exon 7 contributes to Nrp1 binding.A, Glu-154 interacts with the side chain hydroxyl (bond distance = 2.73 Å) and backbone amide (bond distance = 3.16 Å) of Thr-299 of the Nrp1 L1 loop. B, mutation of Glu-154 to alanine (E154A, purple bar) reduces binding to Nrp1 (p < 0.0004). AP-tagged wild-type and mutant VEGF were used at an activity of 25 μmol of pNPP hydrolyzed/min per μl. Error bars, S.D.

FIGURE 5.

Exon 7-encoded residues of VEGF-A164 are responsible for Nrp1 binding selectivity.A, superimposition of Nrp1 (PDB code 1KEX) and Nrp2 b1 domains (PDB code 2QQJ, residues 276–427) reveals a similar overall architecture, root mean square deviation 0.7Å, but unique amino acid composition of the L1 loop, with Nrp1-T299 and Nrp2-D301 highlighted in gold and cyan, respectively. B, chimeric Nrp1, containing the L1 loop of Nrp2 (gold/cyan), loses >75% binding to VEGF-A164 relative to WT Nrp1 (gold) (p < 0.000004). AP-tagged wild-type and mutant Nrp1 were used at an activity of 1 μmol of pNPP hydrolyzed/min per μl. C, VEGF-A120, which lacks exon 7, has essentially the same affinity for Nrp2 (gray dashed line, Kd = 23 nm ± 1 nm) as it does for Nrp1. VEGF-A164 retains exon 7 and has dramatically reduced affinity for Nrp2 (black line, Kd = 150 nm ± 4 nm) compared with Nrp1. D, Nrp2 shows 3-fold higher retention of E154A (purple bar) relative to WT VEGF-A164 (blue bar) (p < 0.0003). AP-tagged wild-type and mutant VEGF were used at an activity of 25 μmol of pNPP hydrolyzed/min per μl. Error bars, S.D.

FIGURE 6.

HBD of VEGF-A is responsible for selective binding to the Nrp1 b1 domain. Exon 8-encoded residues mediate high affinity binding whereas exon 7-encoded residues primarily govern selectivity.