Protein phase separation by the ABC transporter Rv1747 of Mycobacterium tuberculosis
department or school: Science, Faculty of
data provider: DSpace
degree campus: UBCV
creator: Heinkel, Florian
issued: 2020-01-31T08:00:00Z
issued: 2018
related degree: Doctor of Philosophy - PhD
degree grantor: University of British Columbia
description: The Mycobacterium tuberculosis ABC transporter Rv1747 is a putative exporter of cell wall biosynthesis intermediates. Rv1747 has a cytoplasmic regulatory module consisting of two pThr-interacting Forkhead-associated (FHA) domains connected by a conformationally disordered linker with two phospho-acceptor threonines (pThr). In chapter 2, I report the structures of FHA-1 and FHA-2 determined by X-ray crystallography and NMR spectroscopy, respectively. Relative to the canonical 11-strand beta-sandwich FHA domain fold of FHA-1, FHA-2 is circularly permuted and lacking one beta-strand. Nevertheless, the two share a conserved pThr-binding cleft. FHA-2 is less stable and more dynamic than FHA-1, yet binds model pThr peptides with moderately higher affinity (~ 50 uM versus 500 uM equilibrium dissociation constants). Based on NMR relaxation and chemical shift perturbation measurements, when joined within a polypeptide chain, either FHA domain can bind either linker pThr to form intra- and intermolecular complexes. Protein phase separation has been recently shown to be a fundamental mechanism underlying the clustering of some proteins at the eukaryotic cell membrane. In chapter 3, I show that upon multi-site phosphorylation of the linker by several Mtb serine/threonine protein kinases including PknF, the isolated regulatory module readily multimerizes and phase separates into dynamic liquid droplets with diagnostic properties similar to those exhibited by eukaryotic proteins. The process is reversed by the sole Mtb serine/threonine phosphatase PstP. In the absence of phosphorylation, the Rv1747 regulatory module can still undergo phase separation, albeit at higher protein concentrations and into droplets with more fluid properties. This points to a synergy between specific FHA-pThr binding and additional weak association of the ID linker and/or the FHA domains leading to the pre-requisite multivalent interactions for demixing. Droplet formation of the regulatory module was re…
aggregated CHO: https://circle.library.ubc.ca/rest/handle/2429/66289
note: PROTEIN PHASE SEPARATION BY THE ABC TRANSPORTER Rv1747 OF MYCOBACTERIUM TUBERCULOSIS by Florian Heinkel B.Sc., Ludwig-Maximilians-Universität München, 2012 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Genome Science and Technology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) April 2018 © Florian Heinkel, 2018 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Protein Phase Separation by the ABC Transporter Rv1747 of Mycobacterium Tuberculosis submitted by Florian Heinkel in partial fulfillment of the requirements for the degree of Doctor of Philosohpy in Genome Science and Technology Examining Committee: Lawrence McIntosh Co-supervisor Jörg Gsponer Co-supervisor Natalie Strynadka Supervisory Committee Member Steven Plotkin University Examiner Charles Thompson University Examiner Additional Supervisory Committee Members: Franck Duong Supervisory Committee Member iii Abstract The Mycobacterium tuberculosis ABC transporter Rv1747 is a putative exporter of cell wall biosynthesis intermediates. Rv1747 has a cytoplasmic regulatory module consisting of two pThr-interacting Forkhead-associated (FHA) domains connected by a conformationally disordered linker with two phospho-acceptor threonines (pThr). In chapter 2, I report the structures of FHA-1 and FHA-2 determined by X-ray crystallography and NMR spectroscopy, respectively. Relative to the canonical 11-strand b-sandwich FHA domain fold of FHA-1, FHA-2 is circularly permuted and lacking one b-strand. Nevertheless, the two share a conserved pThr-binding cleft. FHA-2 is less stable and more dynamic than FHA-1, yet binds model pThr peptides with moderately higher affinity (~ 50 µM versus 500 µM equilibrium dissociation constants). Based on NMR relaxation and chemical shift perturbation measurements, w…
has type: Thesis/Dissertation
date issued: 2018-09
is shown at: 10.14288/1.0368621
language: eng
degree discipline: Genome Science and Technology
provider: Vancouver : University of British Columbia Library
publisher: University of British Columbia
rights: Attribution-NonCommercial-NoDerivatives 4.0 International
rights URI: http://creativecommons.org/licenses/by-nc-nd/4.0/
scholar level: Graduate
type: Text
identifier URI: http://hdl.handle.net/2429/66289