Structural basis for ligand and heparin binding to neuropilin B domains

Abstract

Neuropilin (Nrp) is a cell surface receptor with essential roles in angiogenesis and axon guidance. Interactions between Nrp and the positively charged C termini of its ligands, VEGF and semaphorin, are mediated by Nrp domains b1 and b2, which share homology to coagulation factor domains. We report here the crystal structure of the tandem b1 and b2 domains of Nrp-1 (N1b1b2) and show that they form a single structural unit. Cocrystallization of N1b1b2 with Tuftsin, a peptide mimic of the VEGF C terminus, reveals the site of interaction with the basic tail of VEGF on the b1 domain. We also show that heparin promotes N1b1b2 dimerization and map the heparin binding site on N1b1b2. These results provide a detailed picture of interactions at the core of the Nrp signaling complex and establish a molecular basis for the synergistic effects of heparin on Nrp-mediated signaling.

Fig. 1.

Structure of the b1b2 fragment of Nrp-1. (A) Domain organization of Nrp with the CUB domains (a1 and a2), coagulation factor domains (b1 and b2), MAM domain (c), transmembrane helix (TM), and intracellular domain (IC) indicated. (B) Ribbon diagram of b1 and b2 domains (green and blue, respectively) with the linker shown in gold. (C) Ribbon diagram of N1b1b2 with residues contributing to the b1b2 interface from b1 (333, 335, 362–368, 370, and 399–401) and b2 (464, 465, 468, 499, 501, 545–551, 577, and 581) shown in red and the linker region (residues 426, 427, 429, and 432) shown in gold. (D) (Left) Surface representation of the N1b1b2 colored as in A. (Upper Right) The interaction surface of the core b1 domain from the perspective of b2 with the core b1/b2 contact surface in red and linker contact surface in yellow. (Lower Right) The interaction surface of b2 as in D Upper Right.

Fig. 2.

Location of the VEGF binding site on N1b1b2. (A) Ribbon diagram of N1b1b2/Tuftsin complex with N1b1b2 colored as in Fig. 1A and Tuftsin shown as blue sticks. (B) Schematic of the interaction between N1b1b2 (red and black) and Tuftsin (blue). The three ligand binding loops form the ligand binding surface on Nrp. The residues directly contacting Tuftsin (red) are conserved in Nrp-1 and Nrp-2, as shown in the sequence alignment among rat, human, and Xenopus Nrps (r, h, and x, respectively). (C) Atomic detail of the interaction between N1b1b2 (green) and Tuftsin shown with the 2F_o − F_c electron density map (pink) contoured at 0.9 σ. Hydrogen bonds between Tuftsin and N1b1b2 are indicated, and the structure of N1b1b2 in the absence of Tuftsin is shown in gray. (D) Mutation of Tuftsin-interacting residues in N1b1b2 (S346A, E348A, T349A) knocks out the ability of His-tagged N1b1b2 to pull down VEGF165 by using an immobilized metal affinity column. Shown are VEGF binding to resin alone (lane 1), wild-type N1b1b2 load (lane 2), wild-type N1b1b2 elute (lane 3), mutant N1b1b2 load (lane 4), mutant N1b1b2 elute (lane 5), and 50% VEGF165 load (lane 6).

Fig. 3.

Heparin (Hep) binds and induces dimerization of N1b1b2. (A) N1b1b2 binds to heparin Sepharose, eluting in ≈500 mM NaCl. (B) Size-exclusion chromatography of N1b1b2 reveals that heparin binding induces apparent dimerization of N1b1b2. This dimerization requires a length of heparin >6-mer. (C) Neuropilin-2, N2b1b2, also binds heparin Sepharose, eluting in ≈400 mM NaCl. (D) Heparin induces dimerization of Nrp-2 in a similar fashion to that of Nrp-1.

Fig. 4.

The tertiary structure of N1b1b2 domains creates a single heparin binding surface. (A) Heparin Sepharose binding of b1b2 mutants, which maps the surface involved in heparin binding. (B) Surface mapping of the residues identified as important for heparin binding using the orientation used in Fig. 1D (Left) and a 90° rotation about a vertical axis (Right). (C) Electrostatic surface map of N1b1b2 in the same orientations shown in B with a schematic rod marking the position of the heparin binding surface based on the observed electropositive surface patch and mutagenesis.

Fig. 5.

Structural model of Nrp representing the interactions at the core of the ligand binding interface. The HBD of VEGF165 directly interacts with the b1 domain via the terminal residues, encoded by exon 8, and also couples heparin and Nrp binding.