Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding

Abstract

Neuropilins (Nrps) are co-receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins.

Figure 1

VEGF does not block Sema3A-induced growth cone collapse of DRG neurons. (A) Images of axon growth cones. Untreated dorsal root ganglia (DRG) have large actin-rich growth cones (arrowheads) that are significantly reduced in number upon addition of Sema3A. Anti-Nrp antibodies were added at 50 μg/ml; Sema3A was added at 2.2 nM. Only anti-panNrp^A blocks Sema3A-mediated collapse. (B) Quantification of Sema3A-induced growth cone collapse. The percentage of collapsed growth cones were calculated by counting collapsed and uncollapsed growth cones (N=4 explants per condition). Error bars represent standard error of the mean.

Figure 2

Summary of the neuropilin crystal structures. (A) The Nrp ectodomain is comprised of tandem CUB (a1a2), tandem coagulation factor V/VIII (b1b2), and one MAM (c) domain. Cartoon representation of the seven crystal structures presented in this report with the resolution limits listed below. Magenta spheres indicate a bound calcium ion in the a2 domain. (B) Sequence alignment of the a1a2b1b2 domains of human Nrp1 and Nrp2. Secondary structure elements refer to the Nrp2 a1a2b1b2 structure (blue, a1; green, a2; yellow, b1; red, b2) and are named according to conventions adopted for the spermadhesin CUB domain (Romero et al, 1997) and the coagulation factor V C2 domain (Macedo-Ribeiro et al, 1999). Residues boxed in blue and yellow delineate the antibody epitopes for anti-panNrp^A and anti-Nrp1^B, respectively. Amino acids highlighted in orange indicate the Ca²⁺-binding site in a2, while residues in red represent a putative Ca²⁺-binding site in the a1 domain. Residues shaded in green highlight the positions of amino acid that when substituted disrupt interactions between Sema3A and Nrp1 (Gu et al, 2002). This alignment was produced with EsPript (Gouet et al, 2003).

Figure 3

Overall domain architecture of neuropilins. (A) Domain organization of Nrp2 (blue, a1; green, a2; yellow, b1; red, b2) in complex with the Fab fragment of anti-panNrp^A (tan, heavy chain; gray, light chain). N-glycosylated residues are indicated in magenta. (B) Ribbon representation of the Nrp a2b1b2 structures; the orange spheres highlight a bound calcium ion. (C) Superposition of the Nrp2/Fab complex from two different crystal forms based on the a2b1b2 domains. Note the poor superposition of the a1 domains (yellow arrows) in comparison to the a2b1b2 region (black arrows). Structure figures were produced with PyMol (http://www.pymol.org).

Figure 4

Molecular details of the neuropilin CUB domains. (A) In the a2b1b2 structures, the a2 domain includes a bound calcium ion (orange). The Nrp a1 (panel C) and a2 domains lack the β1 strand found in spermadhesin. (B) The calcium ion of the Nrp1 a2 domain is coordinated by three negatively charged amino acids, two main-chain carbonyl oxygens, and a water molecule. These interactions are highly conserved among Nrps (Supplementary Figures S2 and S3). (C) (left panel) Amino acids are colored according to the percentage of solvent-accessible surface that is buried at the Nrp2/Fab interface (red, 75–100%; orange, 50–74%; yellow, 25–49%). Anti-panNrp^A is crossreactive for Nrp1 and Nrp2, and 11 of the 14 residues in the structural epitope are identical (black text, identical; white text, nonconserved; asterisks indicate residues in which the side chain points toward the a1 protein core). Residues outlined in green indicate the position of amino acids that when replaced were shown to abrogate binding between Nrp1 and Sema3A (Gu et al, 2002). (right panel) The C_α atoms of these amino-acid substitutions are indicated as green spheres. C_α atoms shaded in purple represent a putative calcium-binding site. (D) The anti-panNrp^A/Nrp2 interface. Nrp2 is shown as a molecular surface with local concentrations of charged residues colored blue (basic) or red (acidic) as calculated by PyMol. The antibody contact residues are dominated by aromatic resides from CDRs H2, H3, and L3.

Figure 5

Features of the Nrp VEGF- and heparin-binding domains. (A) The molecular surface of the rat (PDB code 2ORZ) (Vander Kooi et al, 2007) and human b1b2 crystal structures are colored as described in Figure 4D. Green arrows indicate an acidic groove that is formed by the ‘spikes' in the b1 domain (Supplementary Figure S5); this feature forms the Tuftsin-binding site of rat Nrp1. Yellow arrows indicate the approximate location of the heparin-binding patch. (B) The sequence conservation (green, 100%; yellow, ⩾75%) of the b1b2 domains among 12 Nrps (Supplementary Figure S3) was mapped onto the surface of the human Nrp1 b1b2 structure. Two highly conserved patches are delineated in red. Residues outlined in cyan indicate those residues that contact the Fab in the anti-Nrp1^B-Fab/b1 complex. The a2 domain (tan) is shown by using a superposition of the b1b2 and a2b1b2 structures from Nrp1. (C) Ribbon representation of the anti-Nrp1^B–Fab/b1 complex (yellow, b1; orange, heavy chain; gray, light chain). (D) The anti-Nrp1^B/b1 interface. The b1 domain is depicted as a molecular surface with a green arrow indicating the Tuftsin/VEGF tail-binding groove. Only CDRs H3 and L1 contact b1.

Figure 6

A Nrp2 crystallographic dimer suggests models for VEGF and Sema3 binding. (A) Nrp2 forms a saddle-shaped dimer in both crystal forms of the Nrp2/Fab complex. This figure highlights the Nrp2 a1a2b1b2 domains from the monoclinic crystal form of the Fab complex. The putative VEGF tail-, heparin-, and semaphorin-binding sites are indicated. (B) Potential models of VEGF/Nrp and semaphorin/Nrp complexes based on the Nrp a1-mediated dimer in the crystal structures.