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Structural and functional studies of Ser/Thr kinase signaling in mycobacterium tuberculosis

Westcott, Sarah Louise; (2004) Structural and functional studies of Ser/Thr kinase signaling in mycobacterium tuberculosis. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Protein phosphorylation is the most prevalent post-translational modification used in cellular signaling pathways and the interplay between kinases and phosphatases ensures that signaling events are rapidly reversible. In eukaryotic systems, phosphorylation occurs predominately on serine, threonine, and tyrosine amino acid side-chains, through the activity of Ser/Thr or tyrosine kinases. The discovery that SH2 domains function through specific interactions with phosphotyrosine-containing epitopes revolutionised our view of tyrosine kinase signaling mechanisms, providing a structural basis for understanding the assembly of a variety of signaling complexes. Until recently, Ser/Thr kinases were thought to function through conformational changes induced in their phosphorylated targets. Several families of proteins/domains, however, have now been shown to act through phosphoserine/ phosphothreonine-specific interactions. Most prominent among these are a growing family of signaling modules called FHA (ForkHead Associated) domains. Unlike SH2 domains, FHAs are found in certain bacterial species, including Mycobacterium tuberculosis (Mtb), providing an opportunity for the study of eukaryotic-like signaling cascades in a prokaryotic host organism. This thesis describes a body of crystallographic, biochemical, and biological experiments designed to investigate the molecular basis of bacterial FHA domain function. Oriented peptide library screening has identified high affinity phosphothreonine-containing peptide motifs and binding of these, and a set of peptide variants, has further probed the basis of affinity and sequence specificity of the FHA domains from two Mtb gene products, Rv0020c and Rv1827c. This has been combined with binding measurements on six site-directed Rv0020CD mutants and high resolution X-ray crystal structures of both peptide-bound and free Rv0020CD 4 FHA to allow a detailed description of the protein-peptide interactions of this molecule to be obtained. Finally, putative in vivo binding partners have been identified through the use of GST pull-down experiments from Mtb extracts and subsequent analysis by MALDI-TOF mass spectrometry.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Structural and functional studies of Ser/Thr kinase signaling in mycobacterium tuberculosis
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Pure sciences; Biological sciences; Protein phosphorylation
URI: https://discovery.ucl.ac.uk/id/eprint/10098685
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