Interactions between a Bacterial Tyrosine Kinase and its Cognate Phosphatase --- A Solution NMR Study
Item
-
Title
-
Interactions between a Bacterial Tyrosine Kinase and its Cognate Phosphatase --- A Solution NMR Study
-
Identifier
-
d_2009_2013:d879026df91a:11219
-
identifier
-
11639
-
Creator
-
Temel, Deniz B.,
-
Contributor
-
Ranajeet Ghose
-
Date
-
2012
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Biophysics | Biochemistry | Bacterial Tyrosine Kinase | Bacterial Tyrosine Phosphatase | NMR | Wzb | Wzc
-
Abstract
-
Bacterial tyrosine kinases (BY-kinases) play a central role in a variety of physiological processes in bacterial cells. Most notable among these processes is the formation of antiphagocytic capsule and biofilm for survival under environmental stress conditions. BY-kinases constitute a unique class of prokaryotic enzymes sharing no sequence or structural homology with their eukaryal counterparts. BY-kinases are regulated by eukaryotic-like protein tyrosine phosphatases and several tyrosine kinase/phosphatase pairs, which have been identified in both gram-positive and gram-negative bacterial species.;The Escherichia coli (K12) BY-kinase Wzc is regulated by a cytosolic Low Molecular Weight Protein Tyrosine Phosphatase (LMW-PTP) Wzb through the autophosphorylation/dephosphorylation of five phosphorylatable tyrosine residues (termed the tyrosine cluster, YC) located in the C-terminal tail of the cytosolic catalytic domain of Wzc. The cycling between autophosphorylated form of Wzc and the Wzb-catalyzed dephosphorylated state, rather than the quantitative phosphorylation state of the YC, appears to play a central role in the synthesis and export of the exopolysaccharide, colanic acid. Despite biochemical evidence that Wzb dephosphorylates YC-phosphorylated Wzc, the nature of the interactions between these two enzymes and the detailed regulatory mechanism has not been elucidated. The aim of this research was to identify the structural, dynamic and mechanistic aspects of the regulation of Wzc by Wzb.;We used state-of-the-art solution-state Nuclear Magnetic Resonance (NMR) techniques in order to illuminate the interaction between Wzc and Wzb. We have obtained near-complete resonance assignments of the catalytic domain of Wzc, the first for a BY-kinase. Utilizing these assignments and chemical shift titrations, we demonstrate that Wzb prevents oligomerization of Wzc by occluding its intramolecular interaction surface, that lies on the opposite face to that housing the Wzc catalytic site, thus facilitating the dephosphorylation of the exposed YC. The YC would be buried, and shielded from Wzb, in oligomeric Wzc. Similar chemical shift titrations on Wzb reveals that Wzc docks onto Wzb using a site proximal to the catalytic site of the latter. NMR spin-relaxation measurements confirm this hypothesis in addition to revealing interesting dynamics in the key regulatory elements in Wzc and Wzb.
-
Type
-
dissertation
-
Source
-
2009_2013.csv
-
degree
-
Ph.D.
-
Program
-
Physics
