Elsevier

Cellular Signalling

Volume 25, Issue 12, December 2013, Pages 2702-2708
Cellular Signalling

Crystal structure of Src-like adaptor protein 2 reveals close association of SH3 and SH2 domains through β-sheet formation,☆☆

https://doi.org/10.1016/j.cellsig.2013.08.040Get rights and content

Highlights

  • SLAP2 SH3 and SH2 domains are separated by a short SH3–SH2 connector sequence.

  • The structure of SLAP2 reveals close association of the SH3 and SH2 domains.

  • The SH3/SH2 interface involves β-sheet formation and novel surfaces.

  • Our results suggest the SLAP2 SH3 and SH2 domains function interdependently.

  • SLAP2 SH3 and SH2 domains illustrate adaptability in mediating interactions.

Abstract

The Src-like adaptor proteins (SLAP/SLAP2) are key components of Cbl-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling in hematopoietic cells. SLAP and SLAP2 consist of adjacent SH3 and SH2 domains that are most similar in sequence to Src family kinases (SFKs). Notably, the SH3–SH2 connector sequence is significantly shorter in SLAP/SLAP2 than in SFKs. To understand the structural implication of a short SH3–SH2 connector sequence, we solved the crystal structure of a protein encompassing the SH3 domain, SH3–SH2 connector, and SH2 domain of SLAP2 (SLAP2–32). While both domains adopt typical folds, the short SH3–SH2 connector places them in close association. Strand βe of the SH3 domain interacts with strand βA of the SH2 domain, resulting in the formation of a continuous β sheet that spans the length of the protein. Disruption of the SH3/SH2 interface through mutagenesis decreases SLAP-32 stability in vitro, consistent with inter-domain binding being an important component of SLAP2 structure and function. The canonical peptide binding pockets of the SH3 and SH2 domains are fully accessible, in contrast to other protein structures that display direct interaction between SH3 and SH2 domains, in which either peptide binding surface is obstructed by the interaction. Our results reveal potential sites of novel interaction for SH3 and SH2 domains, and illustrate the adaptability of SH2 and SH3 domains in mediating interactions. As well, our results suggest that the SH3 and SH2 domains of SLAP2 function interdependently, with implications on their mode of substrate binding.

Introduction

Intracellular signal transduction involves the assembly of multi-protein complexes. Adaptor proteins consist of modular domains, such as Src-homology 2 (SH2) and Src-homology 3 (SH3) domains, which mediate protein–protein interactions and facilitate the formation of larger signaling complexes. SH2 domains recognize phosphorylated tyrosine (pTyr) residues in specific sequence contexts, while SH3 domains bind short proline-rich sequences (PxxP) that generally form a polyproline type II (PPII) helix [1]. A subset of adaptor proteins (eg. Nck, Crk, Grb2, and SLAP/SLAP2) are composed entirely of SH2 and SH3 domains, and yet play distinct and important roles in signal transduction due to the binding specificity of their SH2 and SH3 domains [1], [2].

The adaptor proteins SLAP (Src-like adaptor protein) and SLAP2 contain a myristoylated amino-terminal region, adjacent SH3 and SH2 domains, and a unique carboxy-terminal tail [3], [4], [5], [6]. SLAP and SLAP2 have been most intensely studied in the context of lymphocyte signaling [5], [7], [8], [9], [10]. SLAP deficient mice have increased T and B cell receptor levels, accompanied by enhanced signaling and positive selection of thymocytes [11], [12], [13]. Studies in T cell lines showed that both SLAP and SLAP2 constitutively associate with the E3 ubiquitin ligase c-Cbl through their C-terminal region and that their SH2 domain mediates binding to tyrosine phosphorylated proteins including the tyrosine kinase Zap70 and the zeta-chain of T cell receptor (TCRζ) [5], [7], [9], [14]. Overexpression of either SLAP or SLAP2 in T cells leads to decreased levels of Zap70, as well as reduced levels of TCR and CD3 on the cell surface [4], [5], [6], [9], [14], [15]. In addition to its role in lymphocytes, SLAP is required for Cbl mediated down regulation of the granulocyte macrophage colony-stimulating factor receptor and termination of cytokine signaling [16]. Similarly, evidence suggests that SLAP and SLAP2 are important for Cbl-dependent regulation of receptor tyrosine kinase signaling, such as downregulation of Fms-like tyrosine kinase 3 (Flt3) and colony-stimulating factor 1 receptor (CSF-1R) in myeloid cells [17], [18], [19]. While several SLAP/SLAP2 SH2 domain dependent interactions have been defined, SH3 domain binding proteins have yet to be identified.

Sequence alignment indicates that the SH3 and SH2 domains of SLAP and SLAP2 are most similar to those in Src family kinases (SFKs). In SFKs, the SH3 and SH2 domains not only contribute to substrate recruitment through recognition of PxxP or pTyr containing targets, but they are also essential for negative regulation of kinase activity (reviewed in [20]). In autoinhibited SFK structures, the SH3 domain binds the SH2-kinase linker and the SH2 domain binds to the phosphorylated carboxy-tail, such that the SH3 and SH2 domains are positioned on the back side of the kinase domain and stabilize the inactive conformation [21], [22], [23]. While the SH3–SH2 connector sequence and SH3–SH2 coupling is important in the assembly of the SFK autoinhibited state, the SH3 and SH2 domains make minimal inter-domain contacts and exhibit independent folding and binding activity [20], [22], [24], [25], [26], [27]. Upon activation, the SH3–SH2 connector is structurally modified, allowing for significant inter-domain flexibility [20], [26], [27].

One notable difference in the arrangement of the SH3 and SH2 domains in SLAP/SLAP2 compared to SFKs is a shortened connector sequence. To understand the functional significance of this element, we solved the x-ray crystal structure of a protein encompassing the SH3 domain, SH3–SH2 connector, and SH2 domain of SLAP2. Here, we describe how a shortened SH3–SH2 connector in SLAP2 results in close association of its modular domains in a manner not yet observed for SH3 and SH2 domains. Inter-domain binding of the SH3 and SH2 domains appears to be central to SLAP2 protein stability, with potential implications for SLAP2 molecular recognition.

Section snippets

Cloning, strains, expression and purification

Residues 29–193 of WT human SLAP2 were cloned in frame into pETM-30-2 (a modified version of pETM-30 that contains pGEX-2T-TEV/pProEx MCS). These residues, referred to as SLAP2–32, encompass the SH3 domain, SH3–SH2 connector, SH2 domain, and the N-terminal portion of the C-terminal tail. Mutants were generated by QuikChange site-directed mutagenesis. Isolated SH3 (29–93) and SH2 (92–193) domains were cloned in frame into pETM-30-2.

SLAP2–32 constructs were individually transformed into E. coli

The SH3 and SH2 domains of SLAP2 are in close association

Sequence alignment of human SLAP2 with Src family kinases shows high similarity for the SH3 and SH2 domains (54% and 73% for human Hck, respectively); however, the connector sequence between the two domains is shortened in SLAP and SLAP2 (Fig. 1). To understand the impact of a shortened connector on the structure of SLAP2, protein encompassing the SH3 domain, SH3–SH2 connector, SH2 domain, and the N-terminal portion of the C-terminal tail of human SLAP2, herein referred to as SLAP2–32, was

Discussion

The SH3 and SH2 domains of SLAP2 adaptor protein interact with one another in an alternative mode of binding not yet observed in other proteins with tandem SH3–SH2 domain arrangements [21], [26], [34], [37], [38], [39], [41], [43], or in heterodimers involving SH2- and SH3-domain containing proteins [40]. A key feature of the SLAP2 SH3–SH2 interface is the formation of a continuous β-sheet that spans the modular domains, facilitated by a tight β-hairpin between strand βe of the SH3 domain and

Conclusions

The short SH3–SH2 connector in SLAP2 results in close association of its modular domains, involving the formation of β-sheet interactions (Fig. 2). Disruption of the SH3/SH2 interface by mutagenesis decreases the stability of SLAP2, suggesting that the SH3 and SH2 domains of SLAP2 function interdependently (Fig. 3). Interdependence of the domains and presentation of the SH3 and SH2 canonical peptide binding sites on the same face of the protein suggests that the SH3 and SH2 domains of SLAP2 may

Accession numbers

Coordinates and structure factors have been deposited in the Protein Data Bank with accession number 4M4Z.

Acknowledgments

We thank F. Sicheri for the gift of pETM-30-2 and use of crystallization robots and x-ray home source. We thank A. Davidson for critical reading of the manuscript. This work was supported by a Canadian Institutes of Health Research grant (MOP 12859; CJM) and a fellowship from the American Brain Tumor Association (LEW-G).

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    The authors declare no conflict of interest.

    ☆☆

    Contributors: LEW-G performed the experiments and wrote the manuscript; CJM supervised LEW-G and wrote the manuscript. All authors approve the final article.

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