An increasing number of SH3 domain–ligand interactions continue to be described that involve the conserved peptide‐binding surface of SH3, but structurally deviate substantially from canonical docking of consensus motif‐containing SH3 ligands. Indeed, it appears that that the relative frequency and importance of these types of interactions may have been underestimated. Instead of atypical, we propose referring to such peptides as type I or II (depending on the binding orientation) non‐consensus ligands. Here we discuss the structural basis of non‐consensus SH3 ligand binding and the dominant role of the SH3 domain specificity zone in selective target recognition, and review some of the best‐characterized examples of such interactions.

Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication. The genus Alphavirus contains 29 known species that are transmitted by arthropods and include many important pathogens, such as Chikungunya virus (CHKV), which during the past decade has re-emerged to cause massive epidemics of febrile arthralgia around the Indian Ocean. The role of the alphaviral non-structural protein 3 (nsP3) has been linked to RNA replication and disease pathogenesis, but its molecular functions have remained elusive. Here we show that the nsP3s of CHKV as well as Sindbis and Semliki Forest viruses use a conserved proline-rich motif to interact with the Src-homology-3 (SH3) domain of host cell amphiphysins Amph1 and BIN1/Amph2, two adaptor proteins prominently involved in cellular membrane dynamics. We observed a striking re-localization of amphiphysin to alphaviral replication complexes in infected cells, and found that disruption of the amphiphysin SH3 binding motif in nsP3 strongly suppressed virus replication in vitro and attenuated Semliki Forest virus in infected mice. Thus, we conclude that amphiphysins are novel and important host cell factors involved in supporting alphavirus replication.

We have determined the human genome to contain 296 different Src homology‐3 (SH3) domains and cloned them into a phage‐display vector. This provided a powerful and unbiased system for simultaneous assaying of the complete human SH3 proteome for the strongest binding to target proteins of interest, without the limitations posed by short linear peptide ligands or confounding variables of more indirect methods for protein interaction screening. Studies involving three ligand proteins, human immunodeficiency virus‐1 Nef, p21‐activated kinase (PAK)2 and ADAM15, showed previously reported as well as novel SH3 partners with nanomolar affinities specific for them. This argues that SH3 domains may have a more dominant role in directing cellular protein interactions than has been assumed. Besides showing potentially important new SH3‐directed interactions, these studies also led to the discovery of novel signalling proteins, such as the PAK2‐binding adaptor protein POSH2 and the ADAM15‐binding sorting nexin family member SNX30.

EnterohemorrhagicEscherichia coliO157:H7 translocates 2 effectors to trigger localized actin assembly in mammalian cells, resulting in filamentous actin “pedestals.” One effector, the translocated intimin receptor (Tir), is localized in the plasma membrane and clustered upon binding the bacterial outer membrane protein intimin. The second, the proline-rich effector EspF_U(aka TccP) activates the actin nucleation-promoting factor WASP/N-WASP, and is recruited to sites of bacterial attachment by a mechanism dependent on an Asn-Pro-Tyr (NPY₄₅₈) sequence in the Tir C-terminal cytoplasmic domain. Tir, EspF_U, and N-WASP form a complex, but neither EspF_Unor N-WASP bind Tir directly, suggesting involvement of another protein in complex formation. Screening of the mammalian SH3 proteome for the ability to bind EspF_Uidentified the SH3 domain of insulin receptor tyrosine kinase substrate (IRTKS), a factor known to regulate the cytoskeleton. Derivatives of WASP, EspF_U, and the IRTKS SH3 domain were capable of forming a ternary complex in vitro, and replacement of the C terminus of Tir with the IRTKS SH3 domain resulted in a fusion protein competent for actin assembly in vivo. A second domain of IRTKS, the IRSp53/MIM homology domain (IMD), bound to Tir in a manner dependent on the C-terminal NPY₄₅₈sequence, thereby recruiting IRTKS to sites of bacterial attachment. Ectopic expression of either the IRTKS SH3 domain or the IMD, or genetic depletion of IRTKS, blocked pedestal formation. Thus, enterohemorrhagicE. colitranslocates 2 effectors that bind to distinct domains of a common host factor to promote the formation of a complex that triggers robust actin assembly at the plasma membrane.