The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Accession:
Functional site class:
Integrin RGD-type binding sites
Functional site description:
Integrins are cell adhesion-mediating receptors present in all metazoans. Each integrin is composed of one α and one β subunit; in humans, 18 α and 8 β subunits can combine to form 24 different dimers, each with unique ligand specificities. Eight of the human integrins: αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1, α8β1 and αIIbβ3 can recognize ligands with RGD motifs (D'Souza,1991) present in several proteins from humans and pathogens or parasites. RGD fits into a deep groove between the two subunits with the Arg residue contacting the α subunit and the Asp residue coordinating a divalent cation embedded in the β subunit, held in place by the Metal-Ion-Dependent Adhesion Site (MIDAS) (Xiong,2002), while the flanking residues modify specificity and affinity. The Arg can be replaced by other residues in certain ligands. In addition, an NGR motif can spontaneously deamidate into isoDGR (where isoD is an L-Asp residue), creating a functional reverse RGD-like binding motif (Curnis,2006).
ELMs with same func. site: LIG_IBS_1  LIG_Integrin_isoDGR_2  LIG_Integrin_KxxGD_FGGC_5  LIG_Integrin_RGD_1  LIG_Integrin_RGD_TGFB_3  LIG_Integrin_RGDW_4 
ELM Description:
The RGDW variant motif contains the canonical core integrin binding RGD motif extended with a tryptophan residue on the C-terminal side. This tryptophan is able to form a favourable π-π interaction with a tyrosine residue in the integrin β3 subunit, hence providing selectivity to the two β3 containing integrin dimers: αIIbβ3 and αvβ3. For these two dimers, the arginine residue at +1 of the canonical RGD motif can be replaced by lysine without impairing high affinity binding.

This subtype of RGD-like motifs was discovered in disintegrins, proteins found in snake venoms of members of the Viperidae family. In general, disintegrins with this motif bind to integrin αIIbβ3 with a higher affinity compared to αvβ3 (Scarborough,1993) and having lysine in the +1 position (KGDW) improves specificity towards αIIbβ3 (Scarborough,1991). Currently all verified instances of this motif are from disintegrins, however, several human and other proteins contain reasonable candidates of RGDW or KGDW subsequences. Optimizing the RGD-containing loop of fibronectin (a known human integrin ligand) using phage display led to the replacement of the RGDS sequence with RGDW, changing fibronectin from a generic integrin ligand into a αIIbβ3 specific one (Van Agthoven,2014; Adair,2020). In addition, Eptifibatide (sold under the brand name Integrilin), a cyclic hexapeptide that was modelled after the disintegrin barbourin, is a potent platelet aggregation inhibitor binding with high affinity to integrin αIIbβ3 (Springer,2008; 2vdn).
Pattern: [RK]GDW
Pattern Probability: 0.0000032
Present in taxon: Metazoa
Interaction Domains:
o See 18 Instances for LIG_Integrin_RGDW_4
o Abstract
Integrins are metazoan-specific receptors not present in fungi or viridiplantae. All human cells express one or more of the 24 types of dimeric integrins spanning the plasma membrane (Barczyk,2009), which mediate signals between the intracellular space, and neighbouring cells or the extracellular matrix (Takada,2007; Campbell,2011; Hynes,2002). The presence and ratio of various integrins reflect the cell’s function. Eight of the 24 human integrins, which resemble the evolutionarily most ancient metazoan integrins, can recognize RGD and RGD-like sequence patterns in their ligands: components of the extracellular matrix (ECM), cell surface proteins of cells or other extracellular signalling proteins. These interactions are central to regulating tissue integrity and tissue boundary formation (Julich,2015), blood clotting (Hook,2017), angiogenesis (Atkinson,2014) and bone formation (Marie,2014), and regulating nutrient absorption through gastrointestinal motility (Khalifeh-Soltani,2016), amongst other functions.

Due to their central roles in cellular communication, misregulation of integrins is implicated in a wide range of diseases. Several viruses, such as the foot-and-mouth disease virus, HIV, West Nile or HPV-16 (Hussein,2015; Asokan,2006) have RGD-like motifs embedded in their proteins that can attach to integrins on the host cell surface, aiding cell entry. Several other pathogens, including both bacteria and eukaryotes also harbour RGD-like motifs to interface with the host cells. Integrins are also known to be targeted by disintegrins (Calvete,2003), a class of proteins present in venoms of snakes from the Viperidae family, ticks, leeches and other parasites. Disintegrins form the strongest known integrin interactions with typical affinities in the low nanomolar - high picomolar range. In addition to pathogenesis, endogenous integrin misregulation is connected to non-pathogenic conditions including Alzheimer’s (Donner,2016), cystic fibrosis (Reed,2015), autism spectrum disorder and schizophrenia (Lilja,2018). A focal point of therapeutic integrin research is cancer (Seguin,2015), as integrins play pivotal roles in angiogenesis and metastasis. Yet, despite the nearly 80,000 publications on integrins, only a handful of integrin-drugs are available commercially, all targeting RGD-binding integrins. Eptifibatide (an antithrombotic drug), which is a result of semi-rational peptide design, is the only one where the integrin interacting region of a snake venom disintegrin was successfully copied and integrated into a cyclic peptide (Phillips,1997). Apart from Eptifibatide, such efforts have also produced promising anti-cancer drug candidates, such as Cilengitide and peptides developed to slow down neovasculature formation (Corti,2008). Other preliminary results show that integrin antagonists could provide a means against inflammatory diseases (Maiguel,2011), HIV infection (Arthos,2018), or could be used in regenerative medicine (Rocha,2018).

One of the reasons for the complexity of integrin regulation is the sensitivity of the downstream signalling to the structural details of ligand binding. While the core RGD motif is common to a wide range of ligands, the exact structural details of the binding determine if the ligand acts as a full or partial agonist or antagonist. There are four major alterations/additions to the presence of the RGD motif that influence this agonistic/antagonistic behaviour, as well as tuning the affinity of the binding and the selectivity profile of the ligand (i. e. which integrin dimers can it bind to):
- First, the flanking residues of the core RGD motif, especially the residues following the Asp, have a huge influence on selectivity and binding strength. Certain integrins have multiple binding modes and these flanking residues are able to determine which binding mode a given ligand will use. For example αv αvβ6 and αvβ8 integrins can bind ligands where the RGD and the following sequence region are in coil conformation, such as fibronectin. However, the same integrins can also bind ligands where RGD is followed by a short α helix interacting with the β6 or β8 subunit via hydrophobic contacts, such as for TGFβ-1 and -3. The two binding modes require different C-terminal flanking residues and influence the binding strength to the same integrins.
- Second, the Arg residue in RGD can be replaced with other residues, most notably Lys, and it can also have a variable position taking advantage of the different side chain length of Lys compared to Arg. Since the interactions formed by Asp itself can be sufficient for biologically relevant binding, the positive charge of RGD can even be omitted in some functional motif instances.
- Third, integrins can bind their ligands in an inverted orientation using a reverse motif. In this case, the Asp residue has to be replaced by its mirror image pair, namely L-Asp. Under physiological conditions, Asn residues followed by Gly can spontaneously decay into L-Asp via spontaneous deamidation (Corti,2011; Curnis,2010). Hence, NGR sequence regions can transform into isoDGR (where isoD represents L-Asp) and they can be actively converted back to NGR by the enzyme protein-L-isoaspartate(D-aspartate) O-methyltransferase (P22061). Natural ligands harbour either an RGD or an NGR motif and some ligands, such as fibronectin, contain both (Curnis,2006).
- Fourth, functional RGD-like motifs often occur in both disordered and ordered regions of proteins. This is in contrast with the notion that most functional motifs reside in disordered protein segments as they need to structurally adapt to their binding partner. However, RGD-like motifs need to adopt a β-turn like conformation to fit into the binding pocket of integrins, and extended surface loops of ordered domains can effectively mimic this conformation. The ordered/disordered nature of an RGD-like motif can heavily influence its binding affinity. As an ordered motif does not lose much conformational entropy upon binding, RGD motifs achieving extremely low Kd values (such as disintegrins (Arruda Macedo,2015)) are most often part of ordered structure, leading to non-transient binding. In contrast, intrinsically flexible ligands such as osteopontin or nephronectin are often disordered to enable a more transient and reversible interaction.
o 9 selected references:

o 21 GO-Terms:

o 18 Instances for LIG_Integrin_RGDW_4
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
P0DM87 Zinc metalloproteinase-disintegrin stejnitin
VM2_TRIST
462 465 EGKLCREAKGDWNNDYCSGQ TP 1 Trimeresurus stejnegeri (Stejneger"s pit viper)
2 
P22827 Disintegrin barbourin
VM2I_SISMB
51 54 KGTVCRVAKGDWNDDTCTGQ TP 4 Sistrurus miliarius barbouri (Dusky pigmy rattlesnake)
2 
Q7LZI5 Disintegrin ussuristatin-1
VM2I1_GLOUS
49 52 AGTVCRVARGDWNDDKCTGQ TP 1 Gloydius ussuriensis (Ussuri mamushi)
2 
P22828 Disintegrin tergeminin
VM2I_SISTE
51 54 KGTVCRVARGDWNDDTCTGQ TP 2 Sistrurus catenatus tergeminus (Western massasauga)
3 
C0HJM4 Disintegrin simusmin
VM2_CROSM
50 53 KGTVCRPARGDWNDDTCTGQ TP 1 Crotalus simus
2 
P0C7X7 Disintegrin mojastin-2
VM212_CROSS
51 54 KGTVCRPARGDWNDDTCTGQ TP 1 Crotalus scutulatus scutulatus (Mojave rattlesnake)
2 
P31986 Disintegrin lutosin
VM2I_CROOL
51 54 KGTVCRVARGDWNDDTCTGQ TP 2 Crotalus oreganus lutosus
3 
P22826 Disintegrin eristicophin
VM2TI_ERIMA
29 32 AGKVCRVARGDWNNDYCTGK TP 3 Eristicophis macmahoni
3 
P0C6S4 Disintegrin eristostatin
VM2TO_ERIMA
27 30 AGKVCRVARGDWNDDYCTGK TP 2 Eristicophis macmahoni
2 
P68521 Disintegrin durissin
VM2I_CRODD
50 53 KGTVCRPARGDWNDDTCTGQ TP 2 Crotalus durissus durissus
3 
P31982 Disintegrin cerastin
VM2I_CROCC
51 54 KGTVCRVARGDWNDDTCTGQ TP 2 Crotalus cerastes cerastes
3 
J9Z332 Zinc metalloproteinase-disintegrin VMP-II
VM2_CROAD
466 469 KGTACRPARGDWNDDTCTGQ TP 1 Crotalus adamanteus (Eastern diamondback rattlesnake)
2 
P17497 Disintegrin bitistatin
VM2_BITAR
64 67 AGTVCRIARGDWNDDYCTGK TP 4 Bitis arietans (Puff adder)
3 
P0C6B6 Zinc metalloproteinase homolog-disintegrin albolatin
VM2AL_TRIAB
462 465 EGTVCRVAKGDWNDDHCTGQ TP 1 Trimeresurus albolabris (White-lipped tree viper)
2 
Q7LZT4 Disintegrin ussuristatin-2
VM2I2_GLOUS
51 54 EGTVCREAKGDWNDDSCTGQ TP 1 Gloydius ussuriensis (Ussuri mamushi)
2 
Q8IZC6 COL27A1
CORA1_HUMAN
1594 1597 GPKGDKGSRGDWGLQGPRGP U 1 Homo sapiens (Human)
Q8IZJ3 CPAMD8
CPMD8_HUMAN
1533 1536 PASAAEGSRGDWPPADDDDP U 1 Homo sapiens (Human)
O43323 DHH
DHH_HUMAN
221 224 RKGLRELHRGDWVLAADASG U 1 Homo sapiens (Human)
Please cite: ELM — the eukaryotic linear motif resource in 2020. (PMID:31680160)

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