Conformational dynamics of the membrane enzyme lipoprotein signal peptidase (LSPA) and characterization of bicelle membrane mimetics

With the rapid growth of antibiotic resistance, it is imperative that new drugs are developed which target novel pathways. The lipoprotein processing pathway is a novel pathway for antibiotic drug targeting as the enzymes involved, Lgt, LspA, and Lnt, are essential in some organisms including E. coli, S. enterica, M. tuberculosis, and S. coelicolor, and have no mammalian homologs. Lipoproteins are characterized by an N- terminal lipid moiety that serves as a membrane anchor, and serve a wide range of functions including in signal transduction, stress sensing, virulence, cell division, sporulation, nutrient uptake, antibiotic resistance, adhesion, and trigger the activation of host innate immune responses. If lipoproteins are not processed correctly they cannot serve these vital functions and the bacteria will be compromised. Lipoprotein signal peptidase (LspA) is an aspartyl protease that carries out the second step in the lipoprotein processing pathway - cleaving the transmembrane helical signal peptide of lipoproteins after lipidation by Lgt.

Lipoprotein signal peptidase (LspA) has been identified as an antibiotic drug target as it targets a novel pathway, is essential in some bacteria, and does not have a mammalian homolog. The crystal structure of LspA has been determined in complex with the antibiotic globomycin. However, the apo and lipoprotein substrate bound structures of LspA have remained elusive. We propose that there are conformational dynamics of LspA in which the b-cradle and PH domains “open” to allow the substrate to enter the active site, or “close” to hide the charged residues of the active site from the surrounding hydrophobic membrane. This hypothesis is investigated using electron paramagnetic resonance (EPR) studies and molecular dynamics (MD) simulations. This hybrid approach allows for visualization of structures consistent with experimental EPR restraints.

In order to efficiently develop drugs to target novel proteins, it is essential to have a biological, quantitative, reproducible, and high-throughput activity assay to test the effectiveness of the developed therapeutics. An activity assay is currently lacking for LspA. Here, a LspA activity assay is sought that will be used to gain a deeper understanding of the protein’s mechanism, test requirements for LspA activity, and ultimately be used to test the efficiency of inhibitors in future antibiotic development.

Membrane proteins, including LspA, hold a wide variety of essential functions and comprise a large percentage of drug targets. However, in order to study membrane proteins in vitro, a membrane mimetic must be used. This membrane mimic must shield the hydrophobic transmembrane residues of the membrane protein so that it is stable and able to be studied in an aqueous environment. Here, work to better characterize bicelle systems used to study membrane proteins will be described. The classically described bicelle contains a central disk-shaped lipid bilayer encircled by a rim of detergents which screen the hydrophobic lipid tails from water. Characterization of DMPC/DHPC bicelles, including bicelle shape and lipid/detergent mixing, is investigated via SAXS, SANS, MD and fluorescence anisotropy.