Mechanisms of Bacterial Tyrosine Kinase Activation and Regulation

Protein phosphorylation, the covalent modification of target proteins on specific serine, threonine or tyrosine residues through the transfer of the phosphate moiety of ATP is a fundamental process operative in all domains of life and regulates most critical cellular processes. This post-translational modification of substrate proteins is facilitated by biocatalysts called protein kinases.

In bacterial cells, almost all the tyrosine phosphorylation activity is driven by members of the BY-kinase family. BY-kinases are ubiquitous in, and unique to the bacterial domain, and are without any known eukaryotic or archaeal orthologs. These unique protein kinases encode catalytic domains that utilize a P-loop fold characteristic of adenosine triphosphatases, which catalyze the hydrolysis of ATP, in lieu of a canonical dual-lobed “protein kinase fold”.

Not surprisingly, given their unconventional structural features, BY-kinases are predicted to be activated and regulated through mechanisms that are distinct from those that are operative in “conventional” protein kinases. In line with the diversity of phospho-signaling across life’s domains, and the structural and mechanistic evolution of the protein kinases that drive this process, it is the long-term goal of this project to uncover, in atomic detail, the mechanisms of BY-kinase activation and regulation.

Given the role of bacterial tyrosine kinases in virulence and biofilm formation, this project has many potential bioeconomy and human health applications. The broad range of techniques deployed in these studies will facilitate the multi-disciplinary training of a diverse group of scholars who represent the broad demographics of The City College of New York and New York City.

The overall goal of this project is to determine the points of commonality and divergence between the activation mechanisms of BY-kinases encoded by Gram-positive and Gram-negative bacteria. The present studies will utilize the catalytic domain of Escherichia coli (K-12) Wzc and functional constructs of Staphylococcus aureus CapB as prototypical BY-kinases derived from Gram- negative and Gram-positive bacteria, respectively.

A variety of computational approaches will be utilized to generate specific hypotheses that will be validated/tested using several complementary experimental methods including biochemical assays, spin-relaxation measurements of NMR- active nuclei, and quantitative mass spectrometry. This project is supported by the Molecular Biophysics Cluster of the Division of the Molecular and Cellular Biosciences.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.