Structural studies of neuropilin‐2 reveal a zinc ion binding site remote from the vascular endothelial growth factor binding pocket

Abstract

Neuropilin‐2 is a transmembrane receptor involved in lymphangiogenesis and neuronal development. In adults, neuropilin‐2 and its homologous protein neuropilin‐1 have been implicated in cancers and infection. Molecular determinants of the ligand selectivity of neuropilins are poorly understood. We have identified and structurally characterized a zinc ion binding site on human neuropilin‐2. The neuropilin‐2‐specific zinc ion binding site is located near the interface between domains b1 and b2 in the ectopic region of the protein, remote from the neuropilin binding site for its physiological ligand, i.e. vascular endothelial growth factor. We also present an X‐ray crystal structure of the neuropilin‐2 b1 domain in a complex with the C‐terminal sub‐domain of VEGF‐A. Zn2+ binding to neuropilin‐2 destabilizes the protein structure but this effect was counteracted by heparin, suggesting that modifications by glycans and zinc in the extracellular matrix may affect functional neuropilin‐2 ligand binding and signalling activity.

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

“…Especially zinc supplementation was shown to be effective on HIF-1 activity, resulting in lower expression of VEGF [5]. Recently a Zn 2+ site was also identified on the extracellular part of Neuropiline-2, of which the physiological ligand is VEGF-A, and was proposed as a regulatory site [49]. …”

Crystal Structure of the Neuropilin-1 MAM Domain: Completing the Neuropilin-1 Ectodomain Picture

“…In contrast, it is unlikely that the MAM domain of NRP2 would bind calcium; while the residues corresponding to N691 and D796 are conserved, the other two residues from the binding site, E651 and D685, correspond to an asparagine and a threonine, respectively (Figure 3A). Previously, it was reported that the NRP1 a1a2 domains also contain a calcium-binding site (Appleton et al., 2007) and it has recently been shown that the b1 domain of NRP2 has a specific Zn 2+ ion binding site (Tsai et al., 2016). Therefore, the signaling potential of NRPs might be additionally regulated by homeostasis of divalent ions.…”

“…They are composed of five extracellular structural domains, named a1 , at the N terminus, then a2 , b1 , b2 , and c , followed by a single transmembrane helix and a short cytoplasmic tail. Several research groups investigated the detailed atomic structure of the first four extracellular domains, as well as their interaction with biological or synthetic ligands (Lee et al., 2002, Vander Kooi et al., 2007, Appleton et al., 2007, Jarvis et al., 2010, Parker et al., 2012b, Tsai et al., 2016). It has been shown that domains a1 and a2 belong to the structural family of CUB domains and that they are required for interaction with semaphorins (Appleton et al., 2007, Nakamura et al., 1998).…”

“…It has been shown that domains a1 and a2 belong to the structural family of CUB domains and that they are required for interaction with semaphorins (Appleton et al., 2007, Nakamura et al., 1998). NRP domains b1 and b2 share structural homology to coagulation factor V/VIII domains and while both are required for maintaining receptor integrity, series of functional and structural studies have demonstrated that VEGFs interact with the NRPs by binding of the C-terminal ends of VEGFs to the specific site on the b1 domain (Vander Kooi et al., 2007, Tsai et al., 2016). …”

Emerging Roles for Neuropilin-2 in Cardiovascular Disease

“…Despite being 44% identical at the amino acid level, NRP1 and NRP2 exhibit distinct expression patterns and ligand preferences in vivo [ 46 , 55 ]. For example, NRP1 predominantly binds to VEGF-A 165 to mediate angiogenesis [ 56 , 57 , 58 ] in vascular ECs, whereas NRP2 interacts with VEGF-C to regulate lymphangiogenesis [ 46 , 59 , 60 , 61 ].…”