Studies of TomB/YmoB family of proteins: biofilm regulators in enteric bacteria

Abstract

1. Introduction

TomB/YmoB family of proteins was proposed as antitoxins of Hha/YmoA family of proteins. Toxin antitoxin (TA) systems are involved in the regulation of biofilm in bacteria, among other functions. Hha-TomB proteins were related with the regulation of biofilm formation and dispersion.

This PhD thesis was focused on solving the 3D structure of a member of TomB/YmoB family of proteins using Nuclear Magnetic Resonance (NMR), on the realization of functional studies of TomB/YmoB proteins and on the study the oxidation and oligomerization process that seems to control the biological function of the TomB/YmoB family of proteins.

2. Optimization of NMR sample parameters to elucidate the 3D structure of a member of TomB family of proteins by NMR

Compared to other spectroscopic techniques NMR is a relatively insensitive method. To determine the structure of proteins multidimensional experiments require long acquisition times and protein concentrations at the millimolar (mM) range. Optimization of the protein constructs to achieve the required solubility and stability to obtain good quality NMR spectra was the first, non-trivial step for 3D protein structure elucidation. The final conditions of the NMR sample to obtain the 3D-structure of a member from TomB family of proteins were: H6-YmoB-C117S at 1.4mM, pH=7.00 buffered with 20mM [NaH2PO4 + Na2HPO4], 150mM NaCl, 1mM TCEP, 0.1mM EDTA and 0.1% NaN3.

3. Functional studies of TomB family of proteins

Understanding the different mechanisms of biofilm regulation and in particular clarifying the biological activity of TomB family of proteins as antitoxins of Hha in biofilms becomes of primary interest. In order to validate the YmoB constructs used, we also determined the effect of YmoB and [C117S] YmoB in the well-studied biofilm system formed by E. coli. We concluded that; Hha-TomB proteins form a toxin-antitoxin system with Hha the toxin and TomB the antitoxin, in planktonic cells as well as in biofilms, and we also demonstrated that the biological activity of TomB proteins is always related to Hha. We showed that TomB and YmoB are functionally interchangeable in planktonic cells and in biofilms, demonstrating that this TA module is functional in enteric bacteria and we confirmed that the C117S YmoB mutant, our optimal construct for NMR studies, is a biologically active antitoxin of Hha in E. coli planktonic and biofilm systems.

4. 3D Structure determination of [C117S] YmoB by NMR

We obtained a high resolution structure, in which the 20 lowest energy models present an average pairwise RMSD under 1.5Å for the heavy atoms of residues forming α-helices. [C117S] YmoB structure consists in a four helix bundle. The protein is mainly negatively charged, but present two positives patches.

5. Oxidation and oligomerization of YmoB: a structure-driven hypothesis for YmoB function

We observed that YmoB and TomB are labile proteins. Both formed oligomers involving cysteine oxidation. A mutant of YmoB containing a single cysteine residue was relatively stable but even this construct required a special fast NMR data acquisition protocol to determine its three dimensional structure, suggesting that this construct still undergoing slow modifications. The remaining Cys18 and its immediate environment are conserved between YmoB and TomB and C18 is buried in the hydrophobic core of [C117S] YmoB. We hypothesized that this cysteine may be functionally relevant and we investigated the reactivity of this residue and the oligomerization of YmoB. On the basis of the results obtained at least three possibilities exists as to the identity of the active antitoxin: a) the reduced YmoB monomer, b) YmoB dimers (or higher oligomers) and c) YmoB monomer with C18 oxidized as sulphenic acid. Further experiments will be necessary to determine which YmoB identity is the active antitoxin, to elucidate the mechanism of action of TomB/YmoB proteins as antitoxins and to define the exact role of C18 and its capacity to be oxidized into sulphenic acid and thiosulphinate in biofilm regulation.

1. Introducció

Seguint els treballs previs realitzats al laboratori del professor Miquel Pons, aquest projecte s’ha focalitzat en els estudis funcionals i estructurals de la proteïna YmoB. Aquesta proteïna de Yersinia spp. Comparteix una elevada homologia de seqüència amb la proteïna TomB de Escherichia coli. Homòlegs de la família de proteïnes TomB/YmoB estan presents en els bacteris entèrics sense que existeixi homologia de seqüència obvia amb altres famílies de proteïnes ni que es conegui l’estructura tridimensional de cap d’elles.

Així doncs, aquesta tesi doctoral s’ha focalitzat en la obtenció de l’estructura tridimensional mitjançant Ressonància Magnètica Nuclear (RMN) de la proteïna YmoB, en la realització d’estudis funcionals de les proteïnes TomB/YmoB i la valdació funcional de la construcció utilitzada en els experiments de RMN y finalment en l’estudi dels processos d’oxidació cisteínica i oligomeritzacio que semblen regir la funcionalitat biològica de la família de proteïnes TomB/YmoB.

2. Resum dels resultats obtinguts

Els principals resultats obtinguts en aquesta tesi doctoral són:

1. Obtenció d’una mostra óptima per a la realització dels experiments de RMN; mutant [C117S] YmoB a 1.4mM, 20mM de [Na2HPO4 + NaH2PO4], 150mM NaCl, 0.2mM EDTA, 1mM TCEP, 0.01% NaN3, pH=7.00.

2. Demostració de la funció com a Toxina-Antitoxina del parell Hha-TomB, sent Hha la toxina i TomB l’antitoxina, en cèl•lules E. Coli planctòniques i en biofilms. Complementació en E. Coli de la funció com a antitoxina de TomB per part de Ymo i de [C117S] YmoB en cèl•lules planctòniques i en biofilms.

3. Resolució de l’estructura tridimensional de la construcció [C117S] YmoB. La proteïna forma un feix de quatre hèlix i la seva superfície electrostàtica es majoritàriament negativa, mostrant dues àrees ben definides amb càrrega positiva.

4. Identificació de tres possibles espècies de YmoB com a forma activa d’antitoxina; YmoB en la seva forma monomèrica reduïda, dímers (o formes oligomèriques majors) de YmoB y YmoB amb la cisteïna 18 oxidada en forma d’àcid sulfènic.