The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Accession:
Functional site class:
CendR Motif Binding to Neuropilin Receptors
Functional site description:
Neuropilins (NRPs) are essential multifunctional cell surface receptors playing significant roles in various cellular signalling pathways that include VEGF-dependent vascular permeability, semaphorin-dependent axon guidance, angiogenesis, immunity, cell survival, migration, and invasion. NRPs specifically recognize a C-terminal motif, sometimes at a polybasic Furin cleavage site, known as the CendR motif. Physiological ligands such as VEGF-165 and semaphorin 3A interact with the b1 domain of NRP1 and promote cellular internalization. Several viruses such as EBV, HTLV-1, and Lujo also use NRP1 for cellular entry. Recently NRP1 has been identified as an entry point of SARS-CoV-2 via a Furin-generated CendR motif present in the viral S1 protein. Loss of NRP function results in significant cardiovascular and neuronal phenotypes and is also associated with embryonic lethality. Thus NRPs play critical roles in both physiological and pathological contexts and are potential therapeutic targets for viral infection.
ELM Description:
The CendR motif has a C-terminal Arg residue that binds to a cavity on the b1 domain of NRP1 and/or NRP2. Many CendR motifs are exposed upon cleavage by a protease, such as Furin, requiring additional Arg/Lys residues preceding the cleavage site. However, the CendR motifs at uncleaved C-termini are also polybasic, indicating that the basic preference is not just due to the protease site-specificity. The smallest known peptide with a CendR motif is the tetrapeptide Tuftsin TKPR. Of the two coagulation factor domains, the b1 domain contains sites specific for the CendR motif making specific contacts with the terminal Arg, while the b2 domain further stabilizes the interaction. Structures of CendR/NRP b1 domains show a characteristic pattern of three hydrogen bonds between the peptide C-terminus and residues in the NRP1 loop III. The presence of a Pro or Arg at the second position from the carboxyl terminus is considered to increase the affinity of the interaction (Zanuy,2013). Structural studies have shown that in the VEGF-A164: NRP1 complex (4DEQ), Arg190 in the peptide 187KPRR190 contributes the majority of the interaction with NRP1. The guanidinium moiety forms a salt bridge with NRP1 D320 carboxylate oxygens. The C-terminus forms hydrogen bonds with three NRP1-b1 residues, Ser-346, Thr-349, and Tyr-353. Though NRP2 is structurally homologous to NRP1, it binds with reduced affinity as the NRP1 L1 loop Thr-299 is replaced by Asp-301 in the NRP2 L1 loop and that creates electrostatic repulsion with Glu-154 of VEGF A164 (Parker,2012). Another NRP1 ligand, Sema3F also uses the same region for ligand binding and blocks the VEGF-A binding to NRP that leads to the anti-angiogenic activity of Sema3F (Parker,2010).
It is not yet clear if the motif specificity for NRP1 and NRP2 should be represented in ELM by different motif patterns as there is not yet enough experimental data.
Pattern: [RK].{0,2}R$
Pattern Probability: 0.0000428
Present in taxons: Severe acute respiratory syndrome coronavirus 2 Vertebrata Viruses
Interaction Domain:
F5_F8_type_C (PF00754) F5/8 type C domain (Stochiometry: 1 : 1)
o See 12 Instances for LIG_NRP_CendR_1
o Abstract
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).
o 21 selected references:

o 21 GO-Terms:

o 12 Instances for LIG_NRP_CendR_1
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
P0DTC2 S
SPIKE_SARS2
682 685 IGAGICASYQTQTNSPRRAR TP 2 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
1 
Q13275 SEMA3F
SEM3F_HUMAN
778 779 APGAPRSPEPQDQKKPRNRR TP 3 Homo sapiens (Human)
2 
P04202 Tgfb1
TGFB1_MOUSE
275 278 LLMATPLERAQHLHSSRHRR TP 2 Mus musculus (House mouse)
1 
O08665 Sema3a
SEM3A_MOUSE
554 555 AWDGSSCSRYFPTAKRRTRR TP 3 Mus musculus (House mouse)
1 
P49767 VEGFC
VEGFC_HUMAN
226 227 SCRCMSKLDVYRQVHSIIRR TP 3 Homo sapiens (Human)
1 
P23064 env
ENV_HTL1M
309 312 LILPPFSLSPVPTLGSRSRR TP 3 Human T-cell lymphotrophic virus type 1 (isolate MT-2)
1 
Q2F842 ORFV132
Q2F842_ORFV
127 130 KCDCRPRFTTTPPTTTRPPR TP 4 Orf virus
1 
Q00731-2 Vegfa
VEGFA_MOUSE
187 190 CKARQLELNERTCRCDKPRR TP 3 Mus musculus (House mouse)
2 
P15692-4 VEGFA
VEGFA_HUMAN
188 191 CKARQLELNERTCRCDKPRR TP 2 Homo sapiens (Human)
1 
P01858 Phagocytosis-stimulating peptide
TUFT_HUMAN
2 4 TKPR TP 1 Homo sapiens (Human)
1 
Q9GZP0 PDGFD
PDGFD_HUMAN
367 370 DIQLDHHERCDCICSSRPPR TP 4 Homo sapiens (Human)
1 
Q00731-3 Vegfa
VEGFA_MOUSE
143 146 CECRPKKDRTKPEKCDKPRR TP 1 Mus musculus (House mouse)
1 
Please cite: ELM — the eukaryotic linear motif resource in 2020. (PMID:31680160)

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