The Eukaryotic Linear Motif resource for
Functional Sites in Proteins
Functional site class:
SH3 domain ligands
Functional site description:
The SH3 domain is one of the best characterized protein domains. SH3 domains are involved in a wide-range of important cellular processes including intracellular signaling, cytoskeletal rearrangements and cell movement, cell growth and immune responses. They bind to proline-rich sequences with moderate selectivity. Early studies identified “PxxP” as a core conserved sequence motif for SH3 binding. These motifs are referred to as canonical binders, among which class I and class II ligands are distinguished based on their orientation. Since then, SH3 domains recognizing partners with multiple atypical SH3 binding motifs have also been described.
ELMs with same func. site: LIG_SH3_1  LIG_SH3_2  LIG_SH3_3  LIG_SH3_4  LIG_SH3_PxRPPK_7  LIG_SH3_PxxDY_5  LIG_SH3_PxxPPRxxK_8  LIG_SH3_PxxxRxxKP_6 
ELM Description:
In the case of the HPK1-type RxxK motif, the motif core is formed by a PxxP and an RxxK motif in a non-overlapping arrangement PxxPxRxxK (Lewitzky,2004).
The structure of the Mona/Gads C-terminal SH3 domain and the HPK1 peptide complex (1UTI) shows that the N-terminal region of the HPK1 peptide binds differently from that of the canonical RxxK motif (LIG_SH3_PxxxRxxKP_6). In the HPK1 peptide, the PxxP sub-motif forms a PPII helix that precedes the RxxK 310 helix, both being essential for SH3 binding. Alanine scanning experiments, evolutionary conservation patterns and crystal contacts were all considered when establishing the motif pattern (Lewitzky,2004). Based on the complex structure, most residues of the motif from the R-6 position to the R+3 position make extensive contacts with the SH3 binding surface. In the position immediately preceding the PxxP submotif only Pro or small hydrophobic residues can be seen that establish weak hydrophobic contact with the SH3 domain. Only hydrophobic residues Leu, Val and Ile are accepted in the 3rd position of the PxxP submotif because it docks into a hydrophobic pocket of the domain (change to Ala in this position abolished binding (Lewitzky,2004)). In the position between the two submotifs (R-1 position) only Pro can be accepted (Ala mutation weakened the binding (Lewitzky,2004) and indeed only Pro can be seen here in the alignments). Interestingly, unlike in the case of other RxxK motifs, here both R and K can be accommodated at the two positive positions of the RxxK submotif that provides the electrostatic component of binding. These strictly conserved positively charged residues are similarly oriented towards the acidic SH3 surface patches as those in the canonical RxxK motif.
Pattern: [PAVLI]P.[LVI]PP[RK][^P][^P][KR]
Pattern Probability: 4.907e-07
Present in taxon: Vertebrata
Interaction Domain:
SH3 domain (IPR001452) SH3 (src Homology-3) domains are small protein modules containing approximately 50 amino acid residues (Stochiometry: 1 : 1)
o See 6 Instances for LIG_SH3_PxxPPRxxK_8
o Abstract
SH3 (SRC Homology 3) domains constitute one of the largest protein domain families with over 300 representatives in the human proteome and 30 in the yeast proteome. SH3 domains are protein recognition modules that typically function in the assembly of signalosomes and signal transduction (Zarrinpar,2003) in signaling pathways, such as cell growth regulation, endocytosis and remodeling of the cytoskeleton. They are small protein interaction modules consisting of only 60 amino acids. At the secondary structure level, the domain displays a beta-sandwich arrangement of five beta-sheets, 3 loops and a short 310 helix (Saksela,2012). The classical SH3 ligand binding site is made up by two hydrophobic pockets and a negatively charged one usually called the specificity pocket, formed by the RT and the n-Src loops.
SH3 domains generally recognise proline-rich motifs forming poly-proline type helices (PPII helixes) when bound to the SH3 (Aitio,2008). Most of the known and well-studied ligands of SH3 domains have the “PxxP” core motif. The ligands that contain the “PxxP” minimal sequence are now referred to as canonical or typical SH3 binding motifs.
Based on the extensive searches for SH3 binding motifs, a classification system was established where Class I (LIG_SH3_1) and Class II (LIG_SH3_2) ligands were distinguished (Fernandez-Ballester,2004). Because of the pseudo-symmetrical nature of the PPII helix, the PXXP-binding site can recognize peptides in both orientations by using two different binding modes. In both cases the prolines make contact with the two hydrophobic pockets, but the orientation of the peptide will be determined by the position of the charged residue binding to the specificity pocket; the motif description is “(R/K)xxPxxP” for class I and “PxxPx(R/K)” for class II (Aitio,2008). A key conserved surface Trp residue in the SH3 binding pocket is known to adopt two different orientations that, in turn, determine the type of ligand (I or II) specifically recognized by the domain. Interestingly, some of the SH3 domains are capable of binding ligands both in Class I and Class II orientation (Fernandez-Ballester,2004). Interestingly, motif binding by at least a subset of SH3 domains seems to be regulated by receptor tyrosine kinases (RTKs) through phosphorylation of a conserved C-terminal Tyr residue within the domain that disturbs motif binding and thus leads to the collapse of the associated signaling networks (Dionne,2018).
The canonical Class I and II motifs are recognized by diverse SH3 domains. However, a large-scale study on human SH3 domains showed that almost half of the investigated domains exhibit atypical binding specificities with no “PxxP” core (Teyra,2017). The hitherto identified atypical SH3 motifs include the “PxxDY” (LIG_SH3_PxxDY_5) (Li,2005; Kaneko,2008; Saksela,2012), different “RxxK-type” motifs (LIG_SH3_PxxxRxxKP_6; LIG_SH3_PxRPPK_7; LIG_SH3_PxxPPRxxK_8) (Liu,2003; Lewitzky,2001; Harkiolaki,2009; Lewitzky,2004), Px[PA]xPR (LIG_SH3_CIN85_PxpxPR_1) (Kurakin,2003; Rouka,2015), and “RKxxYxxY” (Kang,2000). These atypical recognition specificities are usually associated with only a specific subset of the SH3-containing proteins, which often belong to specific processes/pathways.
“PxxDY'' motif binding has only been identified for a relatively few SH3 domain-containing proteins, including Nck1 (P16333) and Eps8L1 (Q8TE68). Nck1 is an adaptor protein functioning in signal transduction between diverse membrane receptors and the cytoskeleton (Li,2001). During the activation of T-cell receptors (TCRs), Nck1 binds to the “PxxDY'' motif of the CD3ε subunit (32317279). In a phage display screening of 296 human SH3 domains only the Nck1, Nck2, Eps8, Eps8L1, Eps8L3 and Eps8L2 proteins were identified as binders of the motif in Cd3ε, indicating that it is highly specific (Kesti,2007). Besides CD3ε, e3b1/abi-1 and US6NL were also demonstrated to have a functional “PxxDY” motif that binds to the SH3 of Eps8 (Mongiovi,1999). Interestingly, two E.coli effector proteins, namely NleH1 and NleH2, have been identified as Eps8 binders. During infection, probably due to being bound by NleH1 and NleH2, Eps8 shows an altered localization pattern within the cytoplasm that might compromise the formation of new microvilli (Pollock,2022).
RxxK-type SH3-binding motifs are specifically recognized by the second (C-terminal) SH3 domains of GRB2 (P62993) and GRAP2 (also called GADS (O75791)) (Liu,2003; Lewitzky,2001; Harkiolaki,2009; Lewitzky,2004), and the two STAM proteins, STAM1 (Q92783) and STAM2 (O75886) have also been described to bind certain RxxK motifs (Kato,2000). RxxK motif-mediated interactions are typically involved in the signaling of T-cell and B-cell receptors, as well as receptor tyrosine kinases. The RxxK motifs are highly versatile: there is a canonical version “PxxxRxxKP” (LIG_SH3_PxxxRxxKP_6) and some variants where the RxxK is preceded by a PxxP motif in an overlapping “PxRPxK” (LIG_SH3_PxRPPK_7)(Harkiolaki,2009) or non-overlapping “PxxPxRxxK” arrangement (LIG_SH3_PxxPPRxxK_8; Lewitzky,2004). There is also a variant described where the R and K residues are placed further apart “RxxxxK” (Harkiolaki,2009). There is at least one available structure for all these variants, therefore the differences in secondary structures adopted in the bound peptides as well as in the contacts established with the SH3 pockets/residues are well-understood (Harkiolaki,2009). Most known RxxK motif-containing proteins, such as LCP2/SLP-76, GAB1, 2 and 3, B-cell linker protein (BLNK), STAM-binding protein and Ubiquitin carboxyl-terminal hydrolase 8 (mUBPY) employ the canonical RxxK for SH3 binding. Some proteins, such as GABs, employ more than one RxxK type, while, for instance, MAP4K1/HPK1 employs the combined motif “PxxPxRxxK” for binding to the C-terminal SH3 of GADS (Lewitzky,2004).
o 3 selected references:

o 5 GO-Terms:

o 6 Instances for LIG_SH3_PxxPPRxxK_8
(click table headers for sorting; Notes column: =Number of Switches, =Number of Interactions)
Acc., Gene-, NameStartEndSubsequenceLogic#Ev.OrganismNotes
Q07889 SOS1
1302 1311 STSQHIPKLPPKTYKREHTH TP 1 Homo sapiens (Human)
425 434 AGGGLPPELPPRRGKPPPLL TP 1 Homo sapiens (Human)
532 541 CRLLNAPPVPPRSAKPLSTS TP 6 Homo sapiens (Human)
Q92918 MAP4K1
469 478 RELDKPPLLPPKKEKMKRKG TP 1 Homo sapiens (Human)
P70218 Map4k1
467 476 PEPGQPPLVPPRKEKMRGKM TP 10 Mus musculus (House mouse)
O35601 Fyb1
404 413 PAHPPVPSLPPRNIKPPLDL TP 4 Mus musculus (House mouse)
Please cite: The Eukaryotic Linear Motif resource: 2022 release. (PMID:34718738)

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