Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Abstract

Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cellfree expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were cotranslationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY⅐nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.Proteins involved in bacterial cell wall biosynthesis are highly conserved and often essential. In stage I, the cytoplasmic proteins MurA-F are responsible for the formation of the soluble peptidoglycan precursor (Park's nucleotide). In stage II, the integral membrane protein MraY catalyzes the ligation of Park's nucleotide with the membrane-bound undecaprenylphosphate (C 55 -P) 4 resulting into lipid I. The membrane-associated globular protein MurG catalyzes subsequent glycosyl transfer forming lipid II (Fig. 1). As the final monomeric building block in bacterial cell wall biosynthesis, lipid II will be transported through the membrane into the periplasmic space for further polymerization in stage III of cell wall biosynthesis (1).The bacterial cell wall biosynthetic pathway is an important and traditional target for antibiotics and numerous drugs directed against key catalysts for clinical applications have been developed. However, most known drugs such as the penicillin derivatives affect the periplasmic or extracellular steps of peptidoglycan synthesis catalyzed by soluble enzymes. In contrast, almost no clinical drugs have been developed that are targeted against the integral membrane proteins involve...

From Nanodiscs to Isotropic Bicelles: A Procedure for Solution Nuclear Magnetic Resonance Studies of Detergent-Sensitive Integral Membrane Proteins

“…Unassisted helical insertion has been demonstrated to have similar free energies as MP insertion by the Sec61 translocon (Ulmschneider et al, 2014). Furthermore, direct expression into bilayers is gaining recognition as a method to produce functional MPs in lipid bilayers that have never been in contact with detergent (Goren et al, 2009; Henrich et al, 2016; Kalmbach et al, 2007; Long et al, 2012; Yildiz et al, 2012). …”

Nanodiscs in Membrane Biochemistry and Biophysics

“…195 Binding properties of β 1 -adrenergic receptor as a function of lipid composition in Nanodiscs was systematically studied. 196 The same group described the effects of specific lipids on the activity of the MraY translocase 197 and screening for optimal lipid requirements for cell-free expression of membrane proteins with direct incorporation in Nanodiscs. 198 In order to study the effect of various lipids on the conformation and dynamics of multidrug pump LmrP it was assembled in Nanodiscs with PE with a varied degree of methylation of the amino group, specifically DOPE (no methylation), DOPE(Me) 2 (two meth-ylations) and DOPC (fully methylated) and with constant fractions of DOPG (23%) and cardiolipin (10%) in a E.coli polar lipid extract.…”

Insights into the Target Interaction of Naturally Occurring Muraymycin Nucleoside Antibiotics

“…[5] Following a previously described topology model, [6a] the first ligand-free X-ray crystal structure of MraY (from the extremophile Aquifex aeolicus ) confirmed the enzyme to be an integral membrane protein with ten transmembrane helices and five cytoplasmic loops forming the active site. [6b] Mechanistic studies on MraY including mutagenesis have not been fully conclusive.…”

“…All isolated natural products 3 – 8 were initially tested for their inhibitory potency towards two different preparations of MraY: (i) MraY from S. aureus , overexpressed in E. coli and used as crude membranes from overexpressing cells; [5a,10f,12d] and (ii) MraY from Aquifex aeolicus , overexpressed in E. coli and purified to homogeneity in the presence of a non-denaturing detergent (modified protocol from ref. [6b,11a] ).…”