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Structural versatility that serves the function of the HRD motif in the catalytic loop of protein tyrosine kinase, Src.

ABSTRACT: Site-directed mutagenesis is a traditional approach for structure-function analysis of protein tyrosine kinases, and it requires the generation, expression, purification, and analysis of each mutant enzyme. In this study, we report a versatile high throughput bacterial screening system that can identify functional kinase mutants by immunological detection of tyrosine phosphorylation. Two key features of this screening system are noteworthy. First, instead of blotting bacterial colonies directly from Agar plates to nitrocellulose membrane, the colonies were cultured in 96-well plates, and then spotted in duplicate onto the membrane with appropriate controls. This made the screening much more reliable compared with direct colony blotting transfer. A second feature is the parallel use of a protein tyrosine phosphatase (PTP)-expressing host and a non-PTP-expressing host. Because high activity Src mutants are toxic to the host, the PTP system allowed the identification of Src mutants with high activity, while the non-PTP system identified Src mutants with low activity. This approach was applied to Src mutant libraries randomized in the highly conserved HRD motif in the catalytic loop, and revealed that structurally diverse residues can replace the His and Arg residues, while the Asp residue is irreplaceable for catalytic activity.

SUBMITTER: Cui Y

PROVIDER: S-EPMC6371218 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Structural versatility that serves the function of the HRD motif in the catalytic loop of protein tyrosine kinase, Src.

Cui Yixin Y Sun Gongqin G

Protein science : a publication of the Protein Society 20181230 3

Site-directed mutagenesis is a traditional approach for structure-function analysis of protein tyrosine kinases, and it requires the generation, expression, purification, and analysis of each mutant enzyme. In this study, we report a versatile high throughput bacterial screening system that can identify functional kinase mutants by immunological detection of tyrosine phosphorylation. Two key features of this screening system are noteworthy. First, instead of blotting bacterial colonies directly ...[more]

PMID: 30461096

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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.

Dataset Information

Structural versatility that serves the function of the HRD motif in the catalytic loop of protein tyrosine kinase, Src.

Publications

Structural versatility that serves the function of the HRD motif in the catalytic loop of protein tyrosine kinase, Src.

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