Exploring the Conformations of Nonfunctional Variants of the Lipid II Flippase MurJ in Escherichia coli.

Abstract

Biosynthesis of the bacterial peptidoglycan cell wall is essential for determining bacterial shape and protection from differences in osmolaric pressure. One necessary step for the construction of the bacterial cell wall is the translocation of the lipid-linked peptidoglycan precursor, Lipid II, across the cytoplasmic membrane. MurJ is the flippase that translocates lipid II in Escherichia coli anda target of recently discovered antibacterials. Little is known about the mechanism of flippase proteins such as MurJ. However, crystal structures recently have shown MurJ in multiple inward- and outward-open conformations. These crystal structures, along with structure-guided in vivo cysteine cross-linking data, have suggested an alternating-access mechanism of function for MurJ. Since this cross-linking method allows probing of the different conformations of MurJ in vivo, here, we utilized this method to examine how changes to residues critical to MurJ function affect the conformation of the flippase in vivo. Our data suggest that MurJ mutant proteins containing R18E stall or slow down function so they are mostly found in an inward-open conformation, while those containing R24E may slightly favor the outward-open conformation.