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

Abstract

Fyn is a non-receptor protein tyrosine kinase that belongs to a highly conserved kinase family, Src family kinases (SFKs). 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 (FN3) monobodies was affinity selected with the 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, 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.

AlphaScreen HTS and Live-Cell Bioluminescence Resonance Energy Transfer (BRET) Assays for Identification of Tau–Fyn SH3 Interaction Inhibitors for Alzheimer Disease

“…5F). Though this hit rate may seem low, the G9 monobody inhibitor, which has an excellent K D for Fyn SH3 (166 nM) 29 , inhibited the D-Tau–Fyn SH3 interaction to a similar degree. Therefore, the fact that several compounds with distinct chemotypes show activity in this stringent assay that requires cell permeability, resistance to cellular degradation, non-toxicity, and high potency is very promising.…”

Non-immunoglobulin scaffold proteins: Precision tools for studying protein-protein interactions in cancer

“…Their ability to target individual domains within endogenous proteins should allow determination of the role of those domains in protein function and signalling cascades. An example of this is shown by SBPs that have been selected against SH3 and SH2 domains for several members of the Src family of proteins [38][39][40][41][42]. This family of non-receptor tyrosine kinases has nine members, Src, Yes, Fyn, Fgr, Yrk, Lyn, Blk, Hck, and Lck, that contain both SH2 and SH3 binding domains in addition to their kinase domain and an SH4 domain [59].…”

“…Monobody reagents have been identified that bind with high specificity and nM affinities to either the SH3 or the SH2 domains within several family members, notably Src, Fyn, Lyn and Lck, [38][39][40][41][42]. The majority of the SH3-binding reagents contained the classical proline-rich SH3 binding motif xxPxxP whereas the SH2 binding reagents were strongly antagonistic to the substrate pYEEI, but did not show a consensus sequence [38][39][40][41][42]. Interestingly, specificity was greater for the SH3 monobody that lacked the consensus sequence, whilst the binder displaying the highest affinity showed a degree of cross-reactivity to a number of family members, namely Yes, Fyn, Lyn and Lck [41].…”

“…Interestingly, specificity was greater for the SH3 monobody that lacked the consensus sequence, whilst the binder displaying the highest affinity showed a degree of cross-reactivity to a number of family members, namely Yes, Fyn, Lyn and Lck [41]. The most potent SH3 binders for Src, Fyn and Lyn could bind their respective targets in cell lysates [39][40][41], but not all binders altered the function of their target as assessed by kinase assay [38,39]. The utility of these 'non-active' binders was subsequently explored and the most potent of Src SH3 binders, 1F11, which bound with similar affinity to Src intracellular binding partners, was found to preferentially bind activated Src, as the SH3 domain is more exposed in this conformation [38].…”

Generating FN3‐Based Affinity Reagents Through Phage Display

“…Not only have they been utilized for affinity reagent generation, but also to improve protein stability (Pershad, 2012), generate inhibitors (Huang, 2012), provide insight into potential protein binding interactions (Halperin, 2003), and rapidly map transcription factor-DNA interactions (Freckleton, 2009). As the technology continues to develop, the opportunities involving phage display will as well.…”