Purification and functional characterization of phiX174 lysis protein E.
Two classes of bacteriophages, the single-stranded DNA Microviridae and the single-stranded RNA Alloleviviridae, accomplish lysis by expressing "protein antibiotics", or polypeptides that inhibit cell wall biosynthesis. Previously, we have provided genetic and physiological evidence that E, a 91-amino acid membrane protein encoded by the prototype microvirus, varphiX174, is a specific inhibitor of the translocase MraY, an essential membrane-embedded enzyme that catalyzes the formation of the murein precursor, Lipid I, from UDP-N-acetylmuramic acid-pentapeptide and the lipid carrier, undecaprenol phosphate. Here we report the first purification of E, which has been refractory to overexpression because of its lethality to Escherichia coli. Moreover, using a fluorescently labeled analogue of the sugar-nucleotide substrate, we demonstrate that E acts as a noncompetitive inhibitor of detergent-solubilized MraY, with respect to both soluble and lipid substrates. In addition, we show that the E sensitivity of five MraY mutant proteins, produced from alleles selected for resistance to E, can be correlated to the apparent affinities determined by in vivo multicopy suppression experiments. These results are inconsistent with previous reports that E inhibited membrane-embedded MraY but not the detergent-solubilized enzyme, which led to a model in which E functions by binding MraY and blocking the formation of an essential heteromultimeric complex involving MraY and other murein biosynthesis enzymes. We discuss a new model in which E binds to MraY at a site composed of the two transmembrane domains within which the E resistance mutations map and the fact that the result of this binding is a conformational change that inactivates the enzyme.
Zheng
Yi
Y
Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, Texas 77843-2128, USA.
Struck
Douglas K
DK
Young
Ry
R
D000483:Alleles; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001433:Bacteriolysis / Q000235:genetics / Q000502:physiology*; D010584:Bacteriophage phi X 174 / Q000737:chemistry* / Q000502:physiology*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005456:Fluorescent Dyes / Q000378:metabolism; D008969:Molecular Sequence Data; D013379:Substrate Specificity / Q000235:genetics; D014166:Transferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups); D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*
D001426:Bacterial Proteins; C029555:E protein, bacteriophage X174; D005456:Fluorescent Dyes; D014764:Viral Proteins; D014166:Transferases; D017855:Transferases (Other Substituted Phosphate Groups); C079803:mraY protein, Bacteria