Dissertations, Theses, and Capstone Projects

Date of Degree

6-2020

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Ranajeet Ghose

Committee Members

David Jeruzalmi

Ruth Stark

David Cowburn

Alexander Shekhtman

Themis Lazaridis

Subject Categories

Biochemistry, Biophysics, and Structural Biology

Abstract

Bacterial tyrosine kinases (BY-kinases) and their cognate Low Molecular Weight Tyrosine Phosphatases (LMW-PTP) work synergistically to regulate diverse physiological processes in both Gram-negative and Gram-positive bacteria. Of these processes, the most widely recognized role of the kinase/phosphatase pair is the biosynthesis and transport of surface polysaccharides. A process that is key in protecting bacterial cell from a variety of environmental stresses through biofilm formation, and the generation of virulence-determining capsules in pathogenic strains. BY-kinases contain a C-terminal tyrosine rich cluster (Y-cluster) located on the catalytic domain that undergoes auto-phosphorylation and is subsequently dephosphorylated by the cognate LMW-PTPs. This phosphorylation/dephosphorylation cycle rather than individual phosphorylation events has been proposed to drive the synthesis and transport of polysaccharides. Despite considerable genetic and biochemical studies, the precise details of the interaction between a BY-kinase and its cognate LMW-PTP, crucial to fully understand the mechanism of regulation governing the phosphorylation/dephosphorylation cycle that is critical for function, remains elusive.

Using the catalytic domain of the E. coli (K12) BY-kinase Wzc (WzcCD) and its cognate LMW-PTP (Wzb) that are involved in the synthesis of the exopolysaccharide colanic acid as a model kinase/phosphatase pair and utilizing a variety of solution NMR-based approaches, we derive a structural model of their complex. This structure provided atomistic insights into the interactions between a BY-kinase and its cognate LMW-PTP for the very first time. The kinase engages a site that lies distal to the catalytic site of the phosphatase but all phosphorylatable tyrosines of the kinase Y-cluster are able to access the catalytic machinery of the phosphatase.

In addition to providing atomistic insight into the interaction, the structure of the complex also allows the interpretation of the millisecond dynamics that are activated in the phosphatase upon engagement of the kinase. Our results suggest that remote docking events are transiently communicated to the active site of phosphatase likely priming its catalytic machinery for the chemical step. We expect that these studies lay the groundwork for similar detailed analyses of the structure and dynamics for other bacterial BY-kinase/phosphatase pairs to provide a clear view on the function of this unique but widely conserved components in bacterial signaling.

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