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Bacterial tyrosine kinases (BY-kinases) comprise a family of protein tyrosine kinases that are structurally distinct from their functional counterparts in eukaryotes and are highly conserved across the bacterial kingdom. BY-kinases act in concert with their counteracting phosphatases to regulate a variety of cellular processes, most notably the synthesis and export of polysaccharides involved in biofilm and capsule biogenesis. Biochemical data suggest that BY-kinase function involves the cyclic assembly and disassembly of oligomeric states coupled to the overall phosphorylation levels of a C-terminal tyrosine cluster. This process is driven by the opposing effects of intermolecular autophosphorylation, and dephosphorylation catalyzed by tyrosine phosphatases. In the absence of structural insight into the interactions between a BY-kinase and its phosphatase partner in atomic detail, the precise mechanism of this regulatory process has remained poorly defined. To address this gap in knowledge, we have determined the structure of the transiently assembled complex between the catalytic core of the Escherichia coli (K-12) BY-kinase Wzc and its counteracting low–molecular weight protein tyrosine phosphatase (LMW-PTP) Wzb using solution NMR techniques. Unambiguous distance restraints from paramagnetic relaxation effects were supplemented with ambiguous interaction restraints from static spectral perturbations and transient chemical shift changes inferred from relaxation dispersion measurements and used in a computational docking protocol for structure determination. This structure presents an atomic picture of the mode of interaction between an LMW-PTP and its BY-kinase substrate, and provides mechanistic insight into the phosphorylation-coupled assembly/disassembly process proposed to drive BY-kinase function.
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This content will become publicly available on December 30, 2026
Profiling tyrosine kinase substrate recognition using bacterial peptide display and deep sequencing
Tyrosine kinases control a wide range of cell signaling pathways that are central to human physiology, and they are dysregulated in a variety of human diseases, most notably cancers. Our understanding of tyrosine kinase biology hinges upon a clear delineation of their protein substrates. Thus, much effort has been invested into defining the substrate specificities of tyrosine kinases, which is partly driven by recognition of the amino acid sequences surrounding the phospho-acceptor tyrosine residues. Numerous methods have been developed to profile tyrosine kinase sequence recognition, and these approaches have collectively demonstrated that different tyrosine kinases have distinct substrate sequence preferences. Here, we describe one such method that combines bacterial peptide display and deep sequencing to study tyrosine kinase substrate preferences. Our approach enables rapid measurement of relative phosphorylation efficiencies for thousands of peptides simultaneously. The genetically-encoded nature of the peptide libraries used with this method allows for facile and cheap construction of libraries tailored to answer a variety questions. Notably, our approach is compatible with genetic code expansion via Amber codon suppression, which allows for the construction and screening of libraries containing non-canonical amino acids. Importantly, results from this assay correlate strongly with quantitative measurements of enzyme kinetics, they corroborate previously reported tyrosine kinase substrate preferences, and they can reveal new insights into tyrosine kinase substrate specificity.
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- Award ID(s):
- 2441001
- PAR ID:
- 10657100
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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