Dataset Information

Selective Targeting of SH2 Domain-Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies.

ABSTRACT: The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of these monobodies competed with pY ligand binding and showed strong selectivity for either the SrcA (Yes, Src, Fyn, Fgr) or SrcB subgroup (Lck, Lyn, Blk, Hck). Interactome analysis of intracellularly expressed monobodies revealed that they bind SFKs but no other SH2-containing proteins. Three crystal structures of monobody-SH2 complexes unveiled different and only partly overlapping binding modes, which rationalized the observed selectivity and enabled structure-based mutagenesis to modulate inhibition mode and selectivity. In line with the critical roles of SFK SH2 domains in kinase autoinhibition and T-cell receptor signaling, monobodies binding the Src and Hck SH2 domains selectively activated respective recombinant kinases, whereas an Lck SH2-binding monobody inhibited proximal signaling events downstream of the T-cell receptor complex. Our results show that SFK SH2 domains can be targeted with unprecedented potency and selectivity using monobodies. They are excellent tools for dissecting SFK functions in normal development and signaling and to interfere with aberrant SFK signaling networks in cancer cells.

SUBMITTER: Kukenshoner T

PROVIDER: S-EPMC5417323 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Selective Targeting of SH2 Domain-Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies.

Kükenshöner Tim T Schmit Nadine Eliane NE Bouda Emilie E Sha Fern F Pojer Florence F Koide Akiko A Seeliger Markus M Koide Shohei S Hantschel Oliver O

Journal of molecular biology 20170325 9

The binding of Src-homology 2 (SH2) domains to phosphotyrosine (pY) sites is critical for the autoinhibition and substrate recognition of the eight Src family kinases (SFKs). The high sequence conservation of the 120 human SH2 domains poses a significant challenge to selectively perturb the interactions of even the SFK SH2 family against the rest of the SH2 domains. We have developed synthetic binding proteins, termed monobodies, for six of the SFK SH2 domains with nanomolar affinity. Most of th ...[more]

PMID: 28347651

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Project description:Protein kinases are enzymes that catalyze the covalent transfer of the γ-phosphate of an adenosine triphosphate (ATP) molecule onto a tyrosine, serine, threonine, or histidine residue in the substrate and thus send a chemical signal to networks of downstream proteins. They are important cellular signaling enzymes that regulate cell growth, proliferation, metabolism, differentiation, and migration. Unregulated protein kinase activity is often associated with a wide range of diseases, therefore making protein kinases major therapeutic targets. A prototypical system of central interest to understand the regulation of kinase activity is provided by tyrosine kinase c-Src, which belongs to the family of Src-related non-receptor tyrosine kinases (SFKs). Although the broad picture of autoinhibition via the regulatory domains and via the phosphorylation of the C-terminal tail is well characterized from a structural point of view, a detailed mechanistic understanding at the atomic-level is lacking. Advanced computational methods based on all-atom molecular dynamics (MD) simulations are employed to advance our understanding of tyrosine kinase activation. The computational studies suggest that the isolated kinase domain (KD) is energetically most favorable in the inactive conformation when the activation loop (A-loop) of the KD is not phosphorylated. The KD makes transient visits to a catalytically competent active-like conformation. The process of bimolecular trans-autophosphorylation of the A-loop eventually locks the KD in the active state. Activating point mutations may act by slightly increasing the population of the active-like conformation, enhancing the availability of the A-loop to be phosphorylated. The Src-homology 2 (SH2) and Src-homology 3 (SH3) regulatory domains, depending upon their configuration, either promote the inactive or the active state of the kinase domain. In addition to the roles played by the SH3, SH2, and KD, the Src-homology 4-Unique domain (SH4-U) region also serves as a key moderator of substrate specificity and kinase function. Thus, a fundamental understanding of the conformational propensity of the SH4-U region and how this affects the association to the membrane surface are likely to lead to the discovery of new intermediate states and alternate strategies for inhibition of kinase activity for drug discovery. The existence of a multitude of KD conformations poses a great challenge aimed at the design of specific inhibitors. One promising computational strategy to explore the conformational flexibility of the KD is to construct Markov state models from aggregated MD data.

Project description:Chlamydiae are well known for their species specificity and tissue tropism, and yet the individual species and strains show remarkable genomic synteny and share an intracellular developmental cycle unique in the microbial world. Only a relatively few chlamydial genes have been linked to specific disease or tissue tropism. Here we show that chlamydial species associated with human infections, Chlamydia trachomatis and C. pneumoniae, exhibit unique requirements for Src-family kinases throughout their developmental cycle. Utilization of Src-family kinases by C. trachomatis includes tyrosine phosphorylation of the secreted effector Tarp during the entry process, a functional role in microtubule-dependent trafficking to the microtubule organizing center, and a requirement for Src-family kinases for successful initiation of development. Nonhuman chlamydial species C. caviae and C. muridarum show none of these requirements and, instead, appear to be growth restricted by the activities of Src-family kinases. Depletion of Src-family kinases triggers a more rapid development of C. caviae with up to an 800% increase in infectious progeny production. Collectively, the results suggest that human chlamydial species have evolved requirements for tyrosine phosphorylation by Src-family kinases that are not seen in other chlamydial species. The requirement for Src-family kinases thus represents a fundamental distinction between chlamydial species that would not be readily apparent in genomic comparisons and may provide insights into chlamydial disease association and species specificity. Chlamydiae are well known for their species specificity and tissue tropism as well as their association with unique diseases. A paradox in the field relates to the remarkable genomic synteny shown among chlamydiae and the very few chlamydial genes linked to specific diseases. We have found that different chlamydial species exhibit unique requirements for Src-family kinases. These differing requirements for Src-family kinases would not be apparent in genomic comparisons and appear to be a previously unrecognized distinction that may provide insights to guide research in chlamydial pathogenesis.

Project description:BackgroundUpon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood.MethodsKnock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies.ResultsCo-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45-/-/Csk-/- non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation.ConclusionsTaken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR.Major findingsOur data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation. Video Abstract.

Dataset Information

Selective Targeting of SH2 Domain-Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies.

Publications

Selective Targeting of SH2 Domain-Phosphotyrosine Interactions of Src Family Tyrosine Kinases with Monobodies.

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