LIG_SH3_CIN85_PxpxPR_1

Most of the SH3-binding partner motifs match the canonical PxxP minimal consensus sequence which is accommodated on the SH3 surface in two xP dipeptide-binding pockets (LIG_SH3_1; LIG_SH3_2; LIG_SH3_3). As is not unusual for linear motif interactions, SH3-interacting motifs can bind to the domain interaction surface in two opposite orientations, termed class I or class II. The non-canonical binding motif PxpxPR discussed here does not contain the PxxP consensus sequence, thus is unlikely to adopt the typical polyproline type II (PPII) helical conformation and mainly binds in the Class II orientation (Saksela,2012). Being a proline-rich motif, it is very likely to be found in flexible natively disordered polypeptide regions rather than within folded domains, thus being accessible as a binding motif.

This non-canonical SH3 binding motif is used in multiple cellular processes of vertebrate organisms. The adaptor protein CIN85 (Cbl-interacting protein of 85 kDa, also known as SH3KBP1; SH3K1_HUMAN) and its close homologue CD2AP (CD2-associated protein; CD2AP_HUMAN) contain three SH3 domains that bind to proteins containing the proline-rich PxpxPR motif (Kurakin,2003; Rouka,2015) and are the main PxpxPR binding partners. Several studies have shown that a combination of the SH3 domains cooperatively bind to targets more strongly than a single SH3 interaction (Kirsch,2001; Konishi,2006; Sato,2007). The two rather large adaptor proteins CIN85 and CD2AP themselves contain a proline-rich region that is bound by SH3 domains of several kinases and adaptor proteins plus a coiled-coil region that mediates homodimerization (Dikic,2002). CIN85 recruits a variety of proteins, most of them associated with membranes and/or the cytoskeleton (Havrylov,2010). Since CIN85 is able to dimerize and use its SH3 domains simultaneously it can bind multiple molecules of the same protein, thus serving as an adaptor that promotes clustering in different process-dependent contexts such as the control of RTK signalling, actin reorganization, T cell functions, kidney architecture and apoptotic signalling (Kowanetz,2003). The interaction of the PxpxPR containing Cbl proteins with the CIN85 SH3 domains plays multiple roles in RTK down-regulation, endocytosis and degradation (Soubeyran,2002; Kowanetz,2003; Jozic,2005; Havrylov,2010). Furthermore, the c-Cbl CIN85 interaction is involved in vesicle mediated endocytosis that is initiated through the ubiquitination of cell surface receptors by c-Cbl (Kowanetz,2004; Havrylov,2010). PxpxPR motif-harbouring partners such as ARAP1, SEPT9 and SHKBP1 are reported to negatively regulate epidermal growth factor receptor (EGFR) endocytosis and degradation by preventing the association of Cbl with CIN85 (Feng,2011; Diesenberg,2015; Li,2018). CD2AP has similar roles as CIN85 in the regulation of RTKs. They are also known to regulate actin cytoskeleton dynamics through direct binding and regulation of the actin capping protein (LIG_ActinCP_CPI_1; Hutchings,2003; Huber,2018). The PxpxPR-mediated interaction of CD2AP with CD2 is crucial for CD2 clustering. This process mediates T-cell polarization as well as recruitment of CD2 during the formation of an immunological synapse (Ceregido,2013). It is known that CD2AP and CIN85 are themselves also able to interact and can together promote cross-linking of actin filaments into bundles indicating a cytoskeleton modulating activity (Havrylov,2010). Both, CD2AP and CIN85 associate with the cytosolic domain of the T-cell surface protein CD2 upon T-cell activation (Ceregido,2013).

CIN85 and CD2AP are large, modular, multi-domain proteins with several copies of SH3 domains and proline-rich motifs. Due to their multivalent interactions and ability to homo-oligomerize, they are perfect candidates for driving liquid-liquid phase separation (LLPS). Indeed, it has been recently demonstrated that in B cells, CIN85 trimerizes through its C-terminal coiled-coil domain and the resulting trimeric CIN85 molecules associate with multiple copies of PxpxPR motifs within several SLP-65 molecules (Src homology (SH) 2 domain-containing leukocyte protein of 65 kDa, also called B-cell linker protein, BLNK; Q8WV28) that recruite additional CIN85 trimers. The resulting extended network of molecular scaffolds (Kuhn,2016) shows the hallmarks of phase-separated liquid condensates (Wong,2020) and is crucial for the efficient initiation of intracellular signalling upon BCR stimulation.

PAK1 contains a PxIxPR motif that is normally recognized by the ARHGEF7 SH3 domain but can be also bound by the second SH3 domain of CIN85, indicating that isoleucine might also be tolerated in the third position of the motif (Jozic,2005). Motifs with valine in the third position were also reported to be recognized by CIN85 (Haglund,2005). However, the binding to CIN85 is low affinity and it is unsure if this is the relevant binding motif (Saksela,2012) and needs to be confirmed by examining further instances.

Strikingly, in comparison to other SH3 domain binding peptides, binding in class I and class II orientation is simultaneously possible (Jozic,2005; 2BZ8; 2AK5) due to the pseudo-symmetric sequence in c-Cbl (P22681) and Cbl-b (Q13191) (RxPxPxPR). However, binding orientation depends on steric properties of the adaptor protein (Ceregido,2013). The binding affinity is in the low micromolar range for individual SH3 domain-PxpxPR interactions, comparable to affinities of other SH3 domains binding to classical polyproline peptides (Kowanetz,2003). Binding stoichiometry can be 2:1 (two SH3 domains held together by a single pseudo-symmetrical peptide sequence of Cbl (2BZ8; Jozic,2005)), or 1:1 depending on steric and sequential properties.

As shown more recently, the functional SH3 domain-binding motif (PxpxPR) is also involved in hijacking signalling pathways with important functions for pathogen infections, as in Theileria annulata-infected cells (Huber,2018) or in infection by Toxoplasma gondii (Guerin,2017). Furthermore, it can be associated with viral infected cells as shown for the chikungunya virus (Agback,2019), human cytomegalovirus (Rak,2018), Hepatitis C virus (Igloi,2015) and Herpes simplex virus 1 (Liang,2005). The protein TA20980 (Q4UGW0) secreted by Theileria annulata interacts specifically with bovine CD2AP (F1MM14) via its functional SH3 domain-binding motif (Huber,2018). Both, CD2AP and CIN85 are engaged in the invasion mechanism used by the apicomplexan parasite Toxoplasma gondii to force the parasite into the host cell (Guerin,2017). Since CD2AP is known to connect cortical cytoskeleton to cytosolic domains of transmembrane proteins, the recruitment of CD2AP to the RON complex of Toxoplasma gondii allows for physically linking it to the host cytoskeleton (Guerin,2017). In chikungunya virus infection, CD2AP has an impact on the initiation of viral replication (Agback,2019). The motif PxxxPR is found in the intrinsically disordered, hypervariable domain of the NSP3 protein of a subgroup of alphaviruses and binds CD2AP (Mutso,2018), an interaction that has been studied mainly in the chikungunya NSP3 protein (A3RMR8; Agback,2019). Human cytomegalovirus encodes for the pUL135 protein (Q64ET4) which contains five putative PxxxPR SH3 domain-binding motifs to interact with CIN85 (Q96B97) and a KxxPxxP motif to bind Abi-1 (Q8IZP0) to take control over EGFR regulation and trafficking in the infected cell. For reactivation of human cytomegalovirus from latency, pUL135 is required to interact with both CIN85 and Abi-1 (Rak,2018). Hepatitis C virus (HCV) NS5A protein impairs epidermal growth factor (EGF) receptor (EGFR) endocytosis and degradation through its interaction with CD2AP (Igloi,2015), while Herpes simplex virus 1 infected cell protein 0 (ICP0) binds CIN85 and Cbl to mediate the clearance of EGF receptor from cell surfaces by promoting its degradation (Liang,2005).