LIG_NRP_CendR_1

Neuropilins (NRPs) are vertebrate-specific single transmembrane helix plasma membrane co-receptors that enhance responses to several growth factors and other mediators under physiological and pathological conditions. The two Neuropilins, NRP1 and NRP2 share many structural and biological properties. They are mainly expressed in endothelial cells, several other normal cell types, and often in malignant tumour cells (Wild,2012). The diverse functions of NRPs have lead to them being termed pleiotropic co-receptors (Guo,2015). NRP1 is mainly involved in neuronal, angiogenic, and cardiovascular development (Parker,2012), whereas NRP2 has a more restricted role in lymphangiogenesis (Parker,2015). NRPs are best known as the receptors of the class 3 semaphorins (SEMA3), which are involved in axonal guidance in embryonic development (Parker,2010), olfactory system development and neuronal control of puberty (Young,2012), and VEGF family members, which regulate angiogenesis, vasculogenesis and endothelial growth (Parker,2012). Other important interactors include the TGF-β1 receptor and the platelet-derived growth factor receptor (Muhl,2017; Glinka,2008). Several viral coat proteins also use NRP1 receptor-mediated endocytosis for host cell entry (Kusunoki,2018; Wang,2015). Also, an immuno-stimulatory tetrapeptide, Tuftsin (TKPR), is a CendR motif and competes with VEGF165 for NRP1 binding (von Wronski,2006). NRP2 has overlapping binding specificity (it is not yet clear if two different motif entries should be prepared) and mainly binds to VEGF-C that is involved in lymphatic endothelial cell function (Parker,2015).
NRP1 and NRP2 share 44% sequence similarity and identical modular architectures. Both consist of a large N-terminal extracellular region, a single transmembrane helix, and a short cytoplasmic region. The extracellular region contains two CUB domains (a1 and a2) followed by two coagulation domains (b1 and b2) and a MAM domain (Vander Kooi,2007). The coagulation factor domains b1 and b2 contain the high-affinity binding site for heparins, as well as various ligands such as VEGF and semaphorin, which contain the specific region known as the CendR motif (Teesalu,2009). Both b1 and b2 domains possess the typical discoidin family β sandwich fold characterized by an eight‐stranded distorted jellyroll β‐barrel, where a five‐stranded antiparallel β‐sheet pack against a three‐stranded antiparallel β‐sheet (Lee,2003). Of these, the conserved interstrand loops at the tip of the b1 domain contain sites important for CendR motif binding and the b2 domain further stabilizes the interaction. The CendR motif must satisfy the C-end Rule, i.e. the strict requirement for exposure of a conserved C-terminal arginine residue for binding a groove in the b1 domain. The free carboxyl group of the basic residue is important for NRP1 binding; adding another amino acid C-terminal after the arginine residue, or blocking the free carboxyl group by amidation, eliminated the binding and internalizing activity (Teesalu,2009). Ligands such as Semaphorins and spike protein of SARS-CoV-2 contain an internal CendR motif that needs to be cleaved by a polybasic protease such as Furin, an extracellular (and Golgi) membrane-bound host cell protease that exposes and activates the motif (Guo,2015). Studies have shown that mutation in the Furin cleavage site of Sema3A causes a serious inherited genetic disorder known as Kallmann syndrome (Hanchate,2012).
Well-known NRP ligands, VEGF-1, and its isoform VEGF-2 (that lacks exon 7) show different binding affinities for NRP1. VEGF-1 uses regions of both exon 7 (residues, Lys-146, Glu-151, and Glu-154.) and exon 8 (residues, 187KPRR$190) for NRP1 binding but the terminal exon-8 encoded regions are necessary for the high-affinity interaction.
The ability of the CendR motif to induce cellular internalization and tissue penetration via NRP1 mediation makes it a good target for cancer drug delivery, as NRP1 is frequently over-expressed in tumour cells. Moreover, the cargo does not need to be attached to a CendR motif for tissue penetration, the free peptide itself induces tissue permeability in the tumour cells and allows the co-injected drug to extravasate and penetrate tumour cells (Teesalu,2013). Furin-processing of CendR motifs of viral glycoproteins of HTLV-1, EBV (Wang,2015; Kusunoki,2018), and now SARS-CoV-2 help them to bind NRP1 and hence aid viral entry and hijack of the host cell. Thus NRP1 provides a novel therapeutic target for COVID-19 (https://www.biorxiv.org/content/10.1101/2020.06.05.134114v1). NRP1 and integrin signalling are intertwined (Valdembri,2009) and, as integrins are also candidate SARS-CoV-2 receptors (Sigrist,2020), these receptors may be important for COVID-19 pathology of the lungs, where ACE2 is largely absent (Hikmet,2020).
Due to a technical limitation in the ELM database structure, SLiMs in proteins generated at internal cleavage sites are not properly handled. A site is missing in the instance list for EBV gB, residues 430-431, (P03188; Wang,2015) and a second site is missing in HTLV-1 env, residues 91-94, (P23064; Kusunoki,2018).