Solution NMR Structure of the SH3 Domain of Human Caskin1 Validates the Lack of a Typical Peptide Binding Groove and Supports a Role in Lipid Mediator Binding

Abstract

SH3 domains constitute an important class of protein modules involved in a variety of cellular functions. They participate in protein-protein interactions via their canonical ligand binding interfaces composed of several evolutionarily conserved aromatic residues forming binding grooves for typical (PxxP) and atypical (PxxxPR, RxxK, RKxxY) binding motifs. The calcium/calmodulin-dependent serine protein kinase (CASK)-interacting protein 1, or Caskin1, a multidomain scaffold protein regulating the cortical actin filaments, is enriched in neural synapses in mammals. Based on its known interaction partners and knock-out animal studies, Caskin1 may play various roles in neural function and it is thought to participate in several pathological processes of the brain. Caskin1 has a single, atypical SH3 domain in which key aromatic residues are missing from the canonical binding groove. No protein interacting partner for this SH3 domain has been identified yet. Nevertheless, we have recently demonstrated the specific binding of this SH3 domain to the signaling lipid mediator lysophospatidic acid (LPA) in vitro. Here we report the solution NMR structure of the human Caskin1 SH3 domain and analyze its structural features in comparison with other SH3 domains exemplifying different strategies in target selectivity. The key differences revealed by our structural study show that the canonical binding groove found in typical SH3 domains accommodating proline-rich motifs is missing in Caskin1 SH3, most likely excluding a bona fide protein target for the domain. The LPA binding site is distinct from the altered protein binding groove. We conclude that the SH3 domain of Caskin1 might mediate the association of Caskin1 with membrane surfaces with locally elevated LPA content.

Novel Roles of SH2 and SH3 Domains in Lipid Binding

“…Nuclear magnetic resonance experiments on the Caskin1 SH3 domain did not reveal any major structural changes upon lysophosphatidic acid addition but instead revealed a discrete set of amino acids that were affected by the binding ( Figure 4 ). In their latest study, the research group presented a nuclear magnetic resonance-solved structure of the human Caskin1 SH3 domain, which validated the lack of a typical peptide binding groove [ 95 ]. Importantly, compared with the c-Src SH3 domain, the Y + Y and W + P pairs of the peptide binding pocket are replaced by K + L and R + P, respectively [ 95 ].…”

“…In their latest study, the research group presented a nuclear magnetic resonance-solved structure of the human Caskin1 SH3 domain, which validated the lack of a typical peptide binding groove [ 95 ]. Importantly, compared with the c-Src SH3 domain, the Y + Y and W + P pairs of the peptide binding pocket are replaced by K + L and R + P, respectively [ 95 ]. In addition, some of the negatively charged residues in the RT-loop are missing, while the distal-loop possesses an acidic region comprising D326 and D332 [ 95 ].…”

“…Importantly, compared with the c-Src SH3 domain, the Y + Y and W + P pairs of the peptide binding pocket are replaced by K + L and R + P, respectively [ 95 ]. In addition, some of the negatively charged residues in the RT-loop are missing, while the distal-loop possesses an acidic region comprising D326 and D332 [ 95 ]. In the n-Src loop, a repositioning of acidic residues is found.…”

“…In the n-Src loop, a repositioning of acidic residues is found. Mapping of the amino acids involved in lysophosphatidic acid binding to the nuclear magnetic resonance structure of the human Caskin1 SH3 domain provided structural evidence that the proline-rich peptide binding groove was indeed missing and demonstrated that lysophosphatidic acid binding involved a domain region distinct from the peptide binding groove generally shared by SH3 domains [95]. The Caskin1 SH3 domain exchanged some proline-binding amino acids and lost the two PXXP motifbinding grooves.…”

Crystal Structure of Caskin1/CASK complex reveals the molecular basis of the binding specificity of CASK_CAMK domain and its binding partners

“…The two proteins have similar domain organization. Their N-terminus contain Ankyrin Repeats, followed by an SH3 domain [22] for lipid binding, a CASK interaction domain (CID), and a SAM domain tandem [23]. In comparison, the C-terminus is the Proline-rich unstructured region.…”