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Jacobs University Project – Fabio Grassi

Modelling the assembly of efflux pumps

Fellow: Fabio Grassi jacobs_university_logo5

Supervisor: Ulrich Kleinekathöfer (Jacobs University Bremen)

Co-supervisor: Prof. Paolo Ruggerone  (University of Cagliari), Martin K. Pos (Goethe University)

f-grassiThe work performed by Fabio Grassi has been focused on the study of the E. Coli TolC by means of molecular dynamics simulations. TolC is a major component of bacterial efflux systems which pumps out the toxic compounds from the cell interior that inhibits the bacteria growth. Results indicate that thus far unreported residues play a key role in conformational stability and identify ion selective sites. These findings can provide valuable insight into the opening mechanism of TolC, thus improving upon the current understanding of efflux pumps and, therefore, aiding in the design of new antibiotics. In a further collaboration between the European Screening Port IME Fraunhofer institute and both ESRs at Jacobs University we are working on a joint project with electrophysiology, all-atom MD simulation and docking studies on TolC.

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The computational group has expertise in simulating the ion and substrate transport through porins but more importantly in modelling TolC and AcrB (in cooperation with the groups from University of Cagliari and Goethe University). It is still an open question how and why TolC and its homologues assume open conformations upon assembling of the tripartite complex. Our group will complement ongoing experimental work using all-atom MD studies. Molecular-level hypotheses by the experimental partners can be tested and new experiments can be suggested. Attention will be devoted to the assembly of the different efflux pump components beyond simple static docking models and learning from the recent CusAB structure.

Molecular fabio-2dynamics simulations of wild type TolC, as well as of nine mutants, were performed with neutral systems and at [KCl] = 0.1 M and 1 M. The effect of ions was studied both by applying electric fields to the system and by restraining the ions outside the protein. Ions were restrained by applying an energy penalty to movement in one dimension, thus restraining them on a plane perpendicular to the main axis of TolC. The criterion for selecting mutants was based upon existing experimental literature and on collaboration with Dr. Vassiliy Bavro.

Findings based on the analysis of hydrogen bonds and salt bridges confirm the importance of certain key residues, namely R367, D371 and D374, located at the periplasmic end, while others, particularly D162, are shown to be of less significance than hitherto believed. Two residues close to the equatorial domain, R328 and R18, are shown to be consistently involved in two interchain salt bridges, while closer to the tip, the salt bridge E359 – R135 is shown to feature prominently throughout the whole simulation: to our knowledge, this is the first time that the effect of these residue pairs is observed. Lastly, analysis of the trajectories of potassium ions has revealed three ion pockets in the beta barrel close to the junction with the periplasmic domain: these sites could potentially provide a target for a properly designed blocking molecule.

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Work on the AcrA-TolC complex is yet be commenced: the investigation of the mutants which exhibited unexpected behaviour proved to be more prolonged than anticipated, resulting in a delay of the second part of the project.

Figure 1. Left: TolC periplasmic domain intra monomer links. Figure 2 right: TolC periplasmic domain inter monomer links. Figure 3 above: ion pocket sites.