Structural and functional investigation of a molecular regulator involved in bacterial silver resistance using NMR

Like werewolves and vampires, bacteria have a weakness: silver. The antimicrobial properties of this precious metal have extensively been used for thousands of years. Despite this long-standing history and its demonstrated activity against Gram-negative bacteria, the complete bactericidal mode of action of silver remains unclear. Nevertheless, silver misuse can damage the cells and a note of caution is mandatory about its potential toxicity. To counteract the toxic effect of silver, Gram-negative bacteria have developed different resistance mechanisms, including the efficient efflux of the metal out of the cell. The first silver-resistant plasmid pMG101 was isolated from Salmonella strain after the death of patients in the burn ward at the Massachusetts General Hospital. The silver-resistant gene cluster is composed of nine genes: a chemiosmotic efflux pump (SilCBA), an ATPase efflux pump (SilP), a responder and membrane sensor performing two-component transcription regulation (SilRS) and three periplasmic silver-binding proteins SilE, SilF and SilG. 

Our group is interested in understanding the complete mechanism of silver ions eviction through the efflux pump system sil. Until now, we tried to decipher how the interplay between SilB, SilF and SilE proteins contribute to the silver efflux pump mechanism1-5. The next challenge will be the understanding of the role of SilP and particularly its metal binding domains and its interplay with SilG. Indeed, the specific role and exact number of MBDs is still enigmatic in terms of metal uptake or regulatory function. An AlphaFold model of SilP informs us about the nature of the long flexible N-ter (158 AA). It highlights the presence of two presumed MBDs (MBD1, 28His-Asp77; MDB2, 92Val-Ser148, Fig. 1B), each containing three cysteine residues forming together a possible silver ion interaction site. The candidate will produce and purify 15N and 15N/13C labeled MBDs and will use NMR spectroscopy to resolve the structure of the two predicted MBDs of SilP and to investigate the interaction between Ag+ and the two MBDs. The protein SilG possesses the conserved CxCC motif involved in metal interaction. An Alphafold model of the SilG/SilP complex reveals a confident interaction site involving this CxCC motif and a conserved Met residue of SilP in a periplasmic flexible 15 residues loop. This putative exit pathway suggests a silver-dependent transfer from cytoplasm to periplasm. Finally, our project proposes to elucidate the silver transport mechanism from the cytoplasm to the periplasm, by studying the structural and dynamical features of SilP, SilG and their interplay.

The project will be hosted by the analytical science institute located in Lyon/Villeurbanne (France). This new institute comprise around 150 researchers and is among the largest analytical science center in Europe. The thesis project will be developed inside the Biosys group and will mainly make use of NMR and will benefit from the expertise of the group members. A part of the project will be dedicated to the production of isotope labeled proteins. The successful candidate should have completed (or in stage of completion) M.Sc. degree either in biochemistry, structural biology, biology, physical chemistry or related fields. Willingness to learn NMR will be strongly appreciated. 

References 

1. Chabert, V.; Hologne, M.; Seneque, O.; Crochet, A.; Walker, O.; Fromm, K. M., Model peptide studies of Ag+ binding sites from the silver resistance protein SilE. Chem comm 2017, 53 (45), 6105-6108. 

2. Chabert, V.; Hologne, M.; Seneque, O.; Walker, O.; Fromm, K. M., Alpha-helical folding of SilE models upon Ag(His)(Met) motif formation. Chem comm 2018, 54 (74), 10419-10422. 

3. Babel, L.; Nguyen, M.-H.; Mittelheisser, C.; Martin, M.; Fromm, K. M.; Walker, O.; Hologne, M., NMR reveals the interplay between SilE and SilB model peptides in the context of silver resistance. Chem comm 2021, 57 (70), 8726-8729. 

4. Arrault, C.; Monneau, Y. R.; Martin, M.; Cantrelle, F.-X.; Boll, E.; Chirot, F.; Comby Zerbino, C.; Walker, O.; Hologne, M., The battle for silver binding: How the interplay between the SilE, SilF, and SilB proteins contributes to the silver efflux pump mechanism. J Biol Chem 2023, 299 (8). 

5. Monneau, Y.; Arrault, C.; Duroux, C.; Martin, M.; Chirot, F.; Mac Aleese, L.; Girod, M.; Comby-Zerbino, C.; Hagège, A.; Walker, O.; Hologne, M., Structural and dynamical insights into SilE silver binding from combined analytical probes. Phys Chem Chem Phys 2023, 25 (4), 3061-3071. 

The "Institut des Sciences analytiques" is a brand new institute (opened in November 2012) which carry out cutting edge science with over 150 researchers. The successful candidate will benefit from several high field NMR spectrometers (400 and 600MHz), several GPUs based computers dedicated to structure calculations and molecular dynamics combined with national computational infrastructure access. Moreover, the lab is fully equipped with devices dedicated to the production of isotope labeled proteins. The duration of this PhD position is 3 years and will be founded by the French Research Ministry. 

The successful candidate should have completed (or in stage of completion) M.Sc. degree either in structural biology or biochemistry. Willingness to learn NMR and complementary techniques will be strongly appreciated.