Isolation of monobodies that bind specifically to the SH3 domain of the Fyn tyrosine protein kinase

Fyn is a nonreceptor protein tyrosine kinase that belongs to a highly conserved kinase family, Src family kinases. Fyn plays an important role in inflammatory processes and neuronal functions. To generate a synthetic affinity reagent that can be used to probe Fyn, a phage-display library of fibronectin type III monobodies was affinity selected with the Src Homology 3 (SH3) domain of Fyn and three binders were isolated. One of the three binders, G9, is specific in binding to the SH3 domain of Fyn, but not to the other members of the Src family (i.e. Blk, Fgr, Hck, Lck, Lyn, Src and Yes), even though they share 51–81% amino acid identity. The other two bind principally to the Fyn SH3 domain, with some cross-reactivity to the Yes SH3 domain. The G9 binder has a dissociation constant of 166 ± 6 nM, as measured by isothermal titration calorimetry, and binds only to the Fyn SH3 domain out of 150 human SH3 domains examined in an array. Interestingly, although the G9 monobody lacks proline in its randomized BC and FG loops, it binds at the same site on the SH3 domain as proline-rich ligands, as revealed by competition assays. The G9 monobody, identified in this study, may be used as a highly selective probe for detecting and purifying cellular Fyn kinase.

Introduction

While recombinant antibodies continue to be a fruitful source of affinity reagents, there has been a great deal of interest in engineering useful affinity reagents from other scaffolds. Several other scaffold proteins have been engineered into affinity reagents [1]. These include three α-helix bundles of the Z domain of protein A, called affibodies [2], avimers [3], cystine-knot peptides [4], green fluorescent protein [5], lipocalin [6], camelid V_HH [7] and designed ankyrin repeat proteins [8], to name a few. The scaffold we have exploited is the fibronectin type III (FN3) domain, which is 94 amino acids in length, and has a three-dimensional structure similar to that of the immunoglobulin fold. There are over 8000 examples of this domain in GenBank, making it one of the most prevalent domains known. It should be pointed out that the tenth repeat of the domain in fibronectin contains the Arg-Gly-Asp (RGD) motif in one loop, which interacts with the fibronectin receptor on cell surfaces [9]. Protein engineering experiments have shown it is possible to substitute amino acids within five of the six loops without loss of stability [10]; in particular, from libraries of variants with combinatorial peptides within two loops on one side of the domain, one generates libraries of synthetic affinity reagents, termed “monobodies”. Through phage display, RNA display, or yeast display, binding FN3 monobodies have been generated for a variety of targets, such as the estrogen receptor [11], integrin [12], lysozyme [13], a phosphorylated IκBα peptide [14], small ubiquitin-like modifier 4 (SUMO4) [15], streptavidin [16], tumor necrosis factor [17], ubiquitin [18] and vascular endothelial growth factor receptor 2 [19]. In addition, the FN3 domain can be expressed inside eukaryotic cells, where it can fold properly and bind to its target [11]. It is also possible to combine a monobody with a protein interaction module to create affinity reagents that can “clamp” short peptides with picomolar affinity 20, 21. Three advantages of the FN3 domain are that it lacks disulfide bonds, it can be highly overexpressed (≥50 mg/L culture) in Escherichia coli and it is thermally stable (T_m = 88 °C).

Recently, we have screened a phage-display library of monobodies for variants that bind to the Src Homology 3 (SH3) domain [16]. This 60 amino acid domain is involved in protein–protein interactions [22], and is present in many different eukaryotic proteins that are involved in signal transduction [23], cytoskeleton assembly [24] and endocytosis [25]. In mammals, the Src family of protein tyrosine kinases (Fig. 1) plays an important role in cell signaling pathways, by transferring signals from activated receptors to downstream signaling proteins. This family of kinases shares a common architecture that consists of an N-terminal myristic acid, a unique region, a SH3 domain, a Src Homology 2 (SH2) domain, a linker and a C-terminal catalytic kinase domain. Generally, these proteins have two conformations: a catalytically inactive conformation, in which the protein is folded into a ‘closed’ state, due to intramolecular interactions, and a catalytically active conformation, in which the protein is in an extended, ‘open’ state [26]. The Fyn protein has been proposed to play a role in T-cell receptor activation [27], lipid utilization [28], exit from meiotic and mitotic metaphases [29], mast cell signaling [30] and carcinogenesis 31, 32.

This work extends prior efforts in which we identified monobodies to the Src SH3 domain [16], and one of the isolated monobodies was converted into a biosensor of in situ activation of Src in cultured cells [33]. We were interested in extending this approach to other members of the Src family, which consists of Blk, Fgr, Fyn, Hck, Lck, Lyn, Src and Yes [34]. We report the isolation of monobodies that are specific in binding to the Fyn SH3 domain. One of the isolated monobodies, G9, is highly selective and can be potentially used as a biosensor of Fyn kinase activation.