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.semaphorin ͉ Tuftsin ͉ VEGF N europilins (Nrps) are essential cell surface receptors with central roles in both angiogenesis and axon guidance (1-3). During angiogenesis, Nrp directly binds VEGF and functions as a coreceptor for VEGF along with VEGF receptor (VEGFR)-2, one of the three VEGFR tyrosine kinases (3, 4). During neural development, Nrp directly binds semaphorin and functions as a semaphorin coreceptor with members of the plexin family (5). Additionally, interactions with both neural adhesion protein L1 and Nrp-interacting protein (NIP), have been shown to be involved in a variety of other cellular processes (6-8).Nrp plays a stimulatory role in angiogenesis, a process critical for growth of solid tumors (reviewed in refs. 4 and 9-11). Nrp expression is observed in tumor vasculature, and overexpression promotes tumorigenesis in vivo for a variety of solid tumors including pituitary, prostate, breast, and colon cancers (12-15). In contrast, a soluble splice form containing only part of the extracellular domain of Nrp inhibits tumorigenesis (16) as do a number of peptides that block VEGF binding to Nrp (17,18). Recent evidence has also demonstrated a role for Nrp in hematological malignancies. Nrp overexpression is observed in both multiple myeloma and acute myeloid leukemia and, in the latter case, is associated with significantly reduced survival (19,20). Strategies to inhibit Nrp activity are thus being developed as potential antitumor therapies (reviewed in ref. 11).Higher eukaryotes possess two Nrp homologs, Nrp-1 and Nrp-2, which share 44% amino acid sequence identity (1). Nrp extracellular regions are composed of two complement binding CUB domains (a1 and a2) followed by two coagulation factor domains (b1 and b2), a MAM (meprin, A5, ) domain (c1), a single membrane-spanning region, and a short cytoplasmic tail (Fig. 1A) (21, 22). The a1 and a2 domains of Nrp are essential for binding to the core seven-bladed Sema domain of semaphorin as well as contributing to interactions with VEGF (23, 24). The coagulation factor domains b1 and b2 contain the high-affinity binding site for the basic heparin binding domain (HBD) of VEGF165 as well a...

VEGF-A121a binding to Neuropilins – A concept revisited

“…2 of 17 ). NRP1 binding to VEGF-A is isolated to C-terminal portion of VEGF-A165a and VEGF-A165a exon 8a encoded residues are indispensible for NRP1 binding.…”

“…2 of 17 ). Thus, NRP1 binding specificity towards VEGF-A isoforms is regulated by VEGF-A exon 8a-encoded sequence, not exon 7 (supplementary figure 8 of 17 ). In 2012, Parker et al reported a crystal structure of mouse VEGF-A164a exons 7+8a-encoded sequence bound to NRP1.…”

“…18 All NRP1-binding proteins and peptides are found to possess a C-terminal arginine. 17 , 19 - 21 The authors further showed that NRP1 has a C-terminal arginine-binding pocket in the b1 domain. Mutating VEGF's C-terminal exon 8a-encoded arginine (R164) resulted in up to 97% loss in retention of mouse VEGF-A164a by NRP1, thus this residue plays a critical role in VEGF-A/NRP1 interactions (Fig.…”

“…36 The abundant negatively-charged HS/CS chains of endothelial-cell-surface proteoglycans bind VEGF-A165a and, as with HS/CS chains in the extracellular matrix, can differentially regulate accessibility/storage of this isoform (and other isoforms with a heparin-binding domain, with affinities depending on the exon 6–8 combinations 37 and tertiary fold). These HS/CS chains can also modify the ability of VEGF isoforms to bind to VEGFR2 and NRPs, which themselves interact with and colocalize with cell surface HSPGs (VEGFR2 was shown to directly interact with HS chains on HSPGs expressed in endothelial cells via a stretch of residues between D6-D7 of VEGFR2 38-41 ; heparin also binds to VEGFR1 42-44 , NRP1 17 and NRP2 45 independent of VEGFs).…”

Neuropilins

“…NRP soluble fragments and VEGF exons 7/8 mimetics that do so have been described and these studies could be expanded. The availability of B domain crystal structures, for example, the b1 domain of NRP1 62 and the B domain of NRP1, cocrystallized with Tuftsin, 63 offer valuable starting points for rational structure-based inhibitor design. For example, mutations in the B domain that would bind VEGF more tightly might be efficient in sequestering VEGF (Geretti E and Klagsbrun M, unpublished).…”