Biophysical studies of outer membrane proteins for novel applications in antibiotics and cancer

The outer membrane is an important layer that separates Gram-negative bacteria from their environment and helps in protection against harmful substances. This layer exists in Gram-negatives and their descendants in Eukaryotic cells: the Mitochondria. Outer membrane proteins (OMPs) are found in the outer membrane of Gram-negative bacteria as well as the mitochondrial outer membrane (MOM). OMPs are characterized with a three-dimensional structure of a β-barrel which stands for β- sheets connected via turns and loops to take a toroidal geometry. Outer membrane proteins can have a variety of functions, including serving as channels or transporters to allow molecules to pass through the membrane, acting as receptors to receive signals from outside the cell, and playing a role in cell adhesion and biofilm formation. Antibiotic resistance stands as one of the major challenges threatening human being’s health and existence. Resistance against the currently existing and clinically in-use antibiotics is being frequently reported and the lack of a discovery pipeline for novel antibiotics with new modes of action is exacerbating the conditions. The short course of antibiotic administration as well as antibiotics standing as the treatment of last resort are examples of the aforementioned fact. As was declared by WHO at 2017, antibiotic discovery should not be left to the market forces, otherwise we will be missing much needed antibiotics the moment we need them the most. The Barrel Assembly Machinery (BAM) complex, is a five-component multiprotein complex located at the outer membrane of Gram-negative bacteria. BAM is a conserved and essential protein complex which folds and inserts the Outer Membrane Proteins (OMPs) to the outer membrane compartment. Essentiality and unique positioning of BAM at the cellular periphery has made it an interesting target for novel antibiotics. This thesis provides structural and functional studies done via state-of-the-art molecular biophysics techniques to decipher the mode of action of novel antibiotics acting on BAM complex and Polymyxins as a membrane active antibiotic. An integrative structural biology approach including Cryo-Electron Microscopy (Cryo-EM) and Nuclear Magnetic Resonance (NMR) Spectroscopy and various other biophysical techniques have been applied to decipher the mode of action of a number of novel antibiotics. Of huge importance, this thesis demonstrates the very first antibiotic target site which is accessible from the outside of the Gram-negative bacteria. This discovery has the benefit to solve the resistance mechanisms imposed by efflux pumps and enzymatic degradation. In another frame, human voltage dependent anion channel (hVDAC) is the most abundant porin in mitochondrial outer membrane which has multiple functions including metabolite flux across MOM as well as signaling related to the apoptotic cell death. This thesis provides sequence specific assignment of hVDAC-1 in native-like lipid bilayer nanodiscs as well as its characterization via Cryo-EM and other biophysical techniques. This in- turn opens up the opportunity of studying hVDAC-1 interacting with its binding partners such as human hexokinase in atomic resolution which has the potential to be used for drug discovery in cancer.