Date of Degree

9-2022

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Ranajeet Ghose

Committee Members

Kevin H. Gardner

Stevan R. Hubbard

Emilio Gallicchio

Paul Robustelli

Subject Categories

Biochemistry, Biophysics, and Structural Biology

Keywords

Protein Kinases, BY-kinases, P-Loop ATPases, Molecular Dynamics, Regulation, ATP

Abstract

The bacterial tyrosine kinases (BY-kinase) represent a class of membrane-bound enzymes that utilize a cycle of auto-phosphorylation and de-phosphorylation to drive the synthesis and export of exopolysaccharides in both Gram-positive and Gram-negative bacteria. The catalytic domain of BY-kinases utilizes a P-loop nucleoside triphosphatase (NTPase) fold that is commonly used by NTPases and small molecule kinases, being the only protein kinase to do so. In the work presented in this thesis, we aimed to obtain an understanding of the mechanisms of the BY-kinases’ unconventional deployment of P-loop scaffold to phosphorylate on tyrosine. We used the BY-kinase of Escherichia coli (K12) as our model and an array of theoretical and experimental approaches to investigate the regulatory dynamics of BY-kinases. Given that BY-kinases belong to the ancient P-loop family, we analyzed regulatory dynamics of a broad class of P-loop enzymes to test whether features of these dynamics are retained despite structural diversification beyond the catalytic site. Our findings reveal that all P-loop enzymes, including BY-kinases, populate global conformations that are correlated to the relative orientations of conserved catalytic elements. These conformational variations modulate the ability to engage Mg2+ and thereby the associated substrate NTP on the path to a chemistry compatible state. These conformational transitions couple to the overall structure of the specific P-loop enzyme through secondary features unique to its context. In the case of BY-kinases, these secondary features involve the interfaces that facilitate oligomerization, a requirement for tyrosine autophosphorylation in trans. Finally, we relate these regulatory properties of BY-kinases to functional phenotypes reported in the literature and provide specific hypotheses that are amenable to future experimental verification.

Manuscript Version

1

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