# Crystal structure of the multidrug resistance regulator RamR complexed with bile acids

Suguru Yamasaki, Ryosuke Nakashima, Keisuke Sakurai, Sylvie Baucheron, Etienne Giraud, Benoît Doublet, Axel Cloeckaert, Kunihiko A Nishiizumi

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Suguru Yamasaki, Ryosuke Nakashima, Keisuke Sakurai, Sylvie Baucheron, Etienne Giraud, et al. Crystal structure of the multidrug resistance regulator RamR complexed with bile acids. 8. Symposium on Antimicrobial Resistance in Animals and the Environment (ARAE 2019), Jul 2019, Tours, France. poster 51. hal-03356645

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Submitted on 28 Sep 2021

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P51

# Crystal structure of the multidrug resistance regulator RamR complexed with bile acids

Suguru YAMASAKI, Ryosuke NAKASHIMA<fnref n="2" />, Keisuke SAKURAI<fnref n="2" />, Sylvie BAUCHERON<fnref n="3" />, Etienne GIRAUD<fnref n="3" />, Benoît DOUBLET<fnref n="3" />, Axel CLECKAERT<fnref n="3" /> and Kunihiko NISHINO<fnref n="1" />,<fnref n="2" />

During infection, *Salmonella* senses and responds to harsh environments within the host. Persistence in a bile-rich environment is important for *Salmonella* to infect the small intestine or gallbladder and the multidrug efflux system AcrAB-TolC is required for bile resistance. The genes encoding this system are mainly regulated by the *ramRA* locus, which is composed of the divergently transcribed *ramA* and *ramR* genes. The *acrAB* and *tolC* genes are transcriptionally activated by RamA, whose encoding gene is itself transcriptionally repressed by RamR. RamR recognizes multiple drugs; however, the identity of the environmental signals to which it responds is unclear. Here, we describe the crystal structures of RamR in complexes with bile components, including cholic acid and chenodeoxycholic acid, determined at resolutions of 2.0 and 1.8 Å, respectively. Both cholic and chenodeoxycholic acids form four hydrogen bonds with Tyr59, Thr85, Ser137 and Asp152 of RamR, instead of π-π interactions with Phe155, a residue that is important for the recognition of multiple compounds including berberine, crystal violet, dequalinium, ethidium bromide and rhodamine 6G. Binding of these compounds to RamR reduces its DNA-binding affinity, resulting in the increased transcription of *ramA* and *acrAB-tolC*. Our results reveal that *Salmonella* senses bile acid components through RamR and then upregulates the expression of RamA, which can lead to induction of *acrAB-tolC* expression with resulting tolerance to bile-rich environments.

<figure><img src="image_2.png" /><figcaption>Fig. 1. Full structure of the RamR dimer with two molecules of cholic or chenodeoxycholic acid. Both compounds were completely enclosed in the binding pocket of RamR. Both cholic and chenodeoxycholic acids form four hydrogen bonds with RamR</figcaption></figure>

## References

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[2] S. Baucheron, F. Coste, S. Canepa, M.C. Maurel, E. Giraud, F. Culard, B. Castaing, A. Roussel and A. Cloeckaert (2012). *Antimicrob Agents Chemother* 56:942-948.

[3] S. Yamasaki, E. Nikaido, R. Nakashima, K. Sakurai, D. Fujiwara, I. Fujii and K. Nishino (2013). *Nat Commun* 4:2078.

[4] S. Baucheron, K. Nishino, I. Monchaux, S. Canepa, M.C. Maurel, F. Coste, A. Roussel, A. Cloeckaert and E. Giraud E (2014). *J Antimicrob Chemother* 69:2400-2406.

[5] S. Yamasaki, R. Nakashima, K. Sakurai, S. Baucheron, E. Giraud, B. Doublet, A. Cloeckaert and K. Nishino (2019). *Sci Rep* 9:177.

<footnote n="1">¹Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.</footnote>

<footnote n="2">²Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan.</footnote>

<footnote n="3">³ISP UMR 1282, INRA, University of Tours, Nouzilly, France</footnote>