Award details

Structure and function of the bacterial redox sensor RsrA an archetypal ZAS protein

Reference	BB/D522911/1
Principal Investigator / Supervisor	Professor Colin Kleanthous
Co-Investigators / Co-Supervisors	Professor Jennifer Potts
Institution	University of York
Department	Biology
Funding type	Research
Value (£)	231,060
Status	Completed
Type	Research Grant
Start date	01/09/2005
End date	31/08/2008
Duration	36 months

Abstract

Through in vitro studies using purified sigma R and RsrA proteins, the project has 4 major research aims that will for the first time provide a molecular picture as to how RsrA senses disulfide stress, how this impacts on the protein¿s structure and its ability to bind Zn(II) ions and sigma R. 1. Structural studies on RsrA and its complex with sigma R. We will determine the NMR structure of RsrA in its oxidised and reduced states. Although up to 3 disulfides can form in RsrA we have shown that a single critical disulfide trigger between Cys11 and Cys44 causes the protein to restructure and dissociate from sigma R. Previous structural work on RsrA has been hampered by the many cysteines in the protein. Using a mutant in which all other cysteines have been substituted for alanine, we have very recently obtained homogeous preparations of RsrA containing the single Cys11-Cys44 disulfide that yields excellent 15N-edited NMR spectra. We will determine the solution structure of oxidised 13C, 15N-labelled RsrA by heteronuclear multidimensional NMR. Reduction of 15N-RsrA C11, C44 disulfided protein with DTT induces dramatic structural changes, the resulting spectrum suggesting that the structure of reduced RsrA may also be attainable. Using new HPLC purification methods of various Cys-to-Ala mutants, we will ob5ain homogeneous preparations of the reduced, zinc bound RsrA for structure determination by NMR. We will also use heteronuclear NMR to map (through perturbation analysis) the binding site of sigma R. 2. Mutating the sigma R-RsrA interface and biophysical characterisation of mutants. Alanine mutants of sigma R binding residues on RsrA will be made and the affinity for the sigma factor determined by isothermal titration calorimetry. This will validate the identification of the binding site and throw light on the underlying thermodynamics of the association. 3. Zinc modulation of RsrA thiol reactivity and release mechanism. We will determine the redox potential for the critical thiol/disulfide redox couple that causes dissociation of the protein from sigma R and how this is modulated by the bound metal ion. This will be accomplished using different ratio¿s of GSH GSSG and or oxidants of known redox potential. We will use quench-flow and stopped-flow methods to follow oxidant-induced release of Zn and attempt to trap potential mixed-disulfides of oxidants with RsrA, which is a possible route by which the metal dissociates from the protein. 4. Why are some ZAS proteins not responsive to disulfide stress, (i) We will overexpress ZAS proteins which have already been shown not to be responsive to oxidative stress and use EXAFS spectroscopy to determine if the metal ligands are the same as in RsrA; (ii) Mutate RsrA Cys11 to His, by analogy with non-redox active ZAS proteins that have histidine residues instead of cysteine close to this key metal binding site.

Summary

Oxygen is an absolute requirement for aerobic respiration, used by animals and microbes alike for energy generation. However, this comes at a price, which is the toxicity (or stress) associated with too much oxygen and or the by-products of respiration (usually termed oxidative stress). This toxicity takes many forms, the two most prominent being the chemical hydrogen peroxide (leading to so-called peroxide stress) and the formation of disulfide bonds, covalent bonds formed between two cysteine amino acids within proteins in the cytoplasm of cells (so-called disulfide stress). These are very harmful to all cell types; hydrogen peroxide, for example, can chemically alter proteins, lipids and DNA, while the formation of disulfide bonds in proteins such as enzymes can inactivate them. Given the potentially catastrophic consequences of oxidative stress, all forms of aerobic life have evolved systems that can tell the cell which particular type of stress is being experienced and switch-on countermeasures that neutralise the stress. The systems responsible for neutralising a particular oxidative stress have been known for many years. For example, particular classes of enzymes, called catalases, consume hydrogen peroxide. In the case of disulfide bonds, certain types of so-called reducing agents are made, known as thioredoxins and glutaredoxins, that can break disulfide bonds and so restore the affected cysteine amino acids to their normal physiological state. We have much less information on the biological systems that actually detect oxidative stress. This is critical if we are to have a deeper understanding of how cells work but also if we are to exploit oxidative stress in biomedicine; Mycobacterium tuberculosis, the bacterium that causes tuberculosis, is able to survive the oxidative onslaught brought about by the cellular guardians of our bodies (macrophages) by virtue of an efficient oxidative stress detection system. This application will dissect at the molecular level a disulfide stress early warning system present in the soil bacterium Streptomyces coelicolor, which is the same system as that found in Mycobacterium tuberculosis. The reason for studying the system from Streptomyces is that we have much more information already collected on this and so have a head-start in trying to understand how it works. The disulfide stress detection system centres on a protein called RsrA. The grant will investigate the three dimensional structure of RsrA and how it changes during disulfide stress, which is the signal that disulfide stress is occurring. We will also investigate how zinc, a transition metal known to bind RsrA, affects its ability to detect disulfide stress. Finally, we will look at proteins that are similar to RsrA from other bacteria in order to understand why only some of them appear to be responsive to disulfide stress. Through this work we will decipher a key oxidative stress sensing system in biology and so further our understanding of how cells deal with the poisonous effects of aerobic respiration.

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Microbiology, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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