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Goethe University Project – Jenifer Cuesta Bernal

logo_goethe_universitaetIdentification of stable RND protein candidates for crystallization studies

Fellow: Jenifer Cuesta Bernal

Supervisor: Prof. Dr. Klaas Martinus Pos  (Goethe Universität Frankfurt)

Co-supervisor: Paolo Ruggerone (University of Cagliari), Ulrich Kleinekathöfer (Jacobs University)

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Multidrug efflux pumps belonging to the Resistance-Nodulation cell Division (RND) superfamily have been intensively studied using biochemical and structural methods. High-resolution structures of wild-type AcrB from E. coli  and several of its single-site variants have been determined via X-ray crystallography. Latest structural data revealed multiple binding sites for drugs simultaneously within the loose and tight promoters. Insights into the drug binding sites are key to further studies on how to develop inhibitors of the RND component and are important sources for forthcoming computational analysis on the drug efflux mechanisms.

A large number of  RND pumps with different substrate specificity have been reported among pathogen bacteria. We selected 6 RND multidrug efflux transporter genes from S. typhimurium and C. jejuni for proof-of-principle of a streamlined, time-saving and economical approach to identify stable protein candidates to enter the larger production and purification. Twelve constructs are synthesized in E. coli as a GFP fusion protein using the versatile FX-cloning procedure. Expression yield and stability within different detergents are directly visualized in crude E. coli detergent extracts (Figure 1). From the initial 6 cloned genes, we obtained 3 pure and stable RND efflux transporters, of which two produced crystals in the initial crystallization screen (Figures 2 and 3). These results are very promising towards the elucidation of RND efflux transporter structure, which will yield valuable data for the design of new antibiotics/inhibitors in the fight against multiple drug resistance. The data will serve as input for all atom MD in Cagliari.

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Figure 1: Detection of RND-GFP fusion proteins by in gel fluorescence and Western Blot.

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Figure 2: Crystals of S. typhimurium AcrB diffracted to 8 Å resolution

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Figure 3: Crystals of C. jejuni CmeB diffracted to 15  Å resolution

The results obtained are in-line with the objectives. We invested time to establish a stream-lined approach, which in the next months will be extended to RND multidrug efflux transporters from E. aerogenes and K. pneumonia.