Biophysical Society Thematic Meeting | Stockholm 2022

Physical and Quantitative Approaches to Overcome Antibiotic Resistance

Poster Abstracts

4-POS Board 4 TOWARDS THE IDENTIFICATION OF BINDING SITES IN THE NISIN IMMUNITY COMPLEX NISFEG Pablo A Cea 1 ; Julia Gottstein 2 ; Sander H.J. Smits 2 ; Holger Gohlke 1,3 ; 1 Heinrich Heine University Düsseldorf, Institute for Pharmaceutical and Medicinal Chemistry, Düsseldorf, Germany 2 Heinrich Heine University Düsseldorf, Institute of Biochemistry, Düsseldorf, Germany 3 Forschungszentrum Jülich, John von Neumann Institute for Computing (NIC), Jülich Supercomputing Centre (JSC), Institute of Biological Information Processing (IBI-7: Structural Biochemistry) & Institute of Bio- and Geosciences (IBG-4: Bioinformatics), Jülich , Germany Nisin is a potent peptidic antibiotic produced by Lactococcus lactis. The nisin biosynthetic operon also encodes for a set of immunity proteins that protect the producing strain against the toxic effects of its own antibiotic. One of these immunity proteins is the NisFEG complex. NisFEG is a tetrameric ABC transporter belonging to the lantibiotic transporter family LanFEG. It is constituted by the heterodimeric transmembrane region of NisE and NisG and the soluble homodimeric region formed by two NisF chains. The presence of this complex alone can confer protection against nisin, but the structural and mechanistic details of how it works remain elusive. In this work, by combining molecular modeling and molecular dynamics simulations, we aim to uncover the structural basis of nisin recognition and how it is exported in the nisin immunity complex NisFEG.Our results reveal the presence of distinctive clefts located between the interfaces of the transmembrane subunits NisE and NisG. Such clefts are also present in other ABC transporters involved in exporting hydrophobic peptides, but not in transporters mediating the transport of membrane components. To assess if the observed clefts could be involved in nisin transport, we performed co-solvent molecular dynamics simulations using multiple small molecule probes. The probes accumulate specifically in a single cleft of the complex, which strongly suggests that it could act as a binding site for nisin. To further test the relevance of this site, we performed site-directed mutagenesis experiments targeting residues within it. Variants lacking aromatic residues in the putative binding cleft show impaired function compared to variants lacking aromatic residues in the opposite cleft. In conclusion, our results point towards an inter-protein surface cleft in NisFEG as a key region in nisin recognition.

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