Untangling topoisomerase inhibition by protein toxins through cryo-electron microscopy and crystallography

This project should appeal to a motivated student who wants to develop both scientific and also project management skills, preparing them for both commercial and academic research. This project will use X-ray crystallography and cryo-electron microscopy and therefore will be conducted across multiple sites, exposing the student to a larger range of scientific communities and practices than offered by other projects. It should also appeal as the techniques we intend to employ are at the forefront of current molecular and structural biology. Our topic offers an exciting program of work, in a relevant field with excellent job prospects.

Background: Mycobacterium tuberculosis (Mtb) infects one-third of the world’s population and in 2015 accounted for 1.8 million deaths, overtaking HIV as the most deadly human pathogen. We have recently demonstrated how clinically-utilised fluoroquinolone antibiotics (global sales ~$8 bn) inhibit DNA gyrase from Mtb, an essential topoisomerase necessary for DNA replication and maintenance of DNA supercoiling. This work also highlighted how antibiotic resistance is rendering fluoroquinolones ineffective. There is an abundance of alternative naturally-occurring antibiotics, from toxin-antitoxin systems, that can target DNA gyrase. Mtb encodes at least eighty different toxin-antitoxin loci, which perform multiple roles such as protection from antibiotics and controlling dormancy inside host macrophages, by specifically halting cell growth. These loci include homologues of the parDE family, which inhibit DNA gyrase. We propose to study the biochemical and structural basis of DNA gyrase inhibition by ParE toxins. Studying these complexes will generate new ways to inhibit a proven therapeutic target using toxins validated by millennia of bacterial evolution.

Project Aims: In this cross-institution, multi-disciplinary proposal we will use a combination of protein biochemistry and structural biology to investigate topoisomerase inhibition by bacterial toxins. The ParE:gyrase complexes are of sufficient size to be ideal candidates for the newly set-up cryo-electron microscopy facilities at both the Diamond Light Source, to which the supervisory team will have access. Cryo-EM work will be complemented by X-ray crystallographic studies. There will be multiple sub-aims; 1) examine the effect of ParE on DNA gyrase, including supercoiling, supercoil relaxation and decatenation activities and determine whether inhibition is universal across ParE homologues; 2) identify residues governing ParE:gyrase interactions by in vitro binding studies, site-directed mutagenesis and in vivo mutagenesis screening; 3) elucidate the structural nature of gyrase inhibition, through both X-ray crystallography and cryo-EM. This work will answer a long-standing question regarding the specifics of ParE toxicity and utilise cutting-edge techniques to obtain (the as-yet elusive) full gyrase:toxin structural models.

For further information see the website: https://www.dur.ac.uk/biosciences/

To apply:
Please complete the online application form and attach a full CV and covering letter - https://forms.ncl.ac.uk/view.php?id=553440.

Informal enquiries may be made to [Email Address Removed]