Directed Evolution of a Highly Specific FN3 Monobody to the SH3 Domain of Human Lyn Tyrosine Kinase

Abstract

Affinity reagents of high affinity and specificity are very useful for studying the subcellular locations and quantities of individual proteins. To generate high-quality affinity reagents for human Lyn tyrosine kinase, a phage display library of fibronectin type III (FN3) monobodies was affinity selected with a recombinant form of the Lyn SH3 domain. While a highly specific monobody, TA8, was initially isolated, we chose to improve its affinity through directed evolution. A secondary library of 1.2 × 109 variants was constructed and screened by affinity selection, yielding three variants, two of which have affinities of ~ 40 nM, a 130-fold increase over the original TA8 monobody. One of the variants, 2H7, displayed high specificity to the Lyn SH3 domain, as shown by ELISA and probing arrays of 150 SH3 domains. Furthermore, the 2H7 monobody was able to pull down endogenous Lyn from a lysate of Burkitt's lymphoma cells, thereby demonstrating its utility as an affinity reagent for detecting Lyn in a complex biological mixture.

Frontotemporal dementia mutant tau (P301L) locks Fyn in an open, active conformation conducive to nanoclustering

“…The fact that SFKs are broadly expressed and share a common negative regulatory mechanism based on intramolecular interactions involving the SH2:tail and SH3:linker has motivated extensive research in exploiting these properties, to design strategies based on the creation of synthetic SH2/SH3-binding small molecules to finely control the activity of SFKs (Huang et al, 2016;Kukenshoner et al, 2017;Moroco et al, 2014;Yadav & Miller, 2007). Interestingly, monobodies (Huang et al, 2016;Huang et al, 2012) and peptoid-based ligands (Li & Lawrence, 2005) that are highly selective for the Fyn SH3 domain have already been generated to modulate the activity of Fyn kinase. We used a mEos2-tagged version of the G9 monobody created specifically to selectively bind Fyn SH3 domain (Huang et al, 2012).…”

“…The SH3 domain is also involved in the interaction of Fyn with its substrates such as the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) (Amaya et al, 2018), p85a (Morton et al, 1996), the palmitoyl-acyl transferase DHHC5 (Brigidi et al, 2015), tau (Lee et al, 1998;Reynolds et al, 2008) and P301L mutant tau (Bhaskar et al, 2005). As displacement of the SH3 domain has been used to activate Fyn molecules and other SFKs Due to the importance of the SH3:helix linker interaction in stabilising the closed conformation of SFKs, several strategies have been developed to manipulate the activity of these enzymes based on the creation of small molecules that selectively bind the SH3 domain (Huang et al, 2016;Kukenshoner et al, 2017;Moroco et al, 2014;Yadav & Miller, 2007). The G9 monobody was specifically generated to interact with the SH3 domain, displaying high specificity for Fyn among other kinases (Huang et al, 2012).…”

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].…”