Minimal requirements for inhibition of MraY by lysis protein E from bacteriophage ΦX174

Abstract

The DNA phage ΦX174 encodes the integral membrane protein E whose expression leads to host cell lysis by inhibition of the peptidoglycan synthesis enzyme MraY. Here we use mutagenesis to characterize the molecular details of the E lysis mechanism. We find that a minimal 18-residue region with the modified wild-type sequences of the conserved transmembrane helix of E is sufficient to lyse host cells and that specific residues within and at the boundaries of this helix are important for activity. This suggests that positioning of the helix in the membrane is critical for interactions with MraY. We further characterize the interaction site of the transmembrane helix with MraY demonstrating E forms a stable complex with MraY. Triggering cell lysis by peptidoglycan synthesis inhibition is a traditional route for antimicrobial strategies. Understanding the mechanism of bacterial cell lysis by E will provide insights into new antimicrobial strategies using re-engineered E peptides.

Fig. 1

ΦX174 lysis protein E and a model of its lysis mechanism. A. Alignment of representative sequences of E isoforms available on NCBI. Five sequences were chosen based on diversity. Residue conservation for all E isoforms above 95%, 85%, and 75% are highlighted in red, orange, and yellow, respectively. The transmembrane (TM) domain is shown in a grey box above. Secondary structure was predicted based on the sequence alignment using Jpred (Cole et al., 2008) and indicated by light-blue box for α-helix and a green arrow for β-strand. Previous mutational studies are shown below the alignment: Non-lytic mutations in red; slow lysis-onset mutations in orange; mutations with no effect in lysis activity in black (Witte et al., 1997, Zheng et al., 2008b, Yu et al., 2011). B. Topology of E and MraY based on the prediction by the TMHMM server. Sequences are overlaid. For MraY, E resistant mutations are shown in green circles while predicted catalytic aspartate residues are shown in yellow circles. Colored circles on E are based on Fig. 2 indicating the effect of alanine mutation. C. Model for the proposed lysis mechanism of E. MraY is active. E inserts into the cytoplasmic membrane with the help of the chaperone SlyD. MraY is inactive when E is bound. The soluble C-terminal domain of E can be replaced with another protein to retain lysis activity.

Fig. 2

Alanine-scanning of the N-terminal domain of E. A. Representative growth curves of alanine-scanning mutants in Lemo21 cells. Lines are based on the average curve from Fig. S2. Expression of each E construct was induced at time= 0. Various phenotypes and protein expression levels of each mutation are categorized by color: black, no obvious change in lysis onset; blue, faster lysis onset due to higher protein levels; lightgreen, delayed lysis onset due to lower protein levels; purple, colony-to-colony variation in lysis onset; orange, delayed lysis onset with no obvious protein level change; red, no lytic activity with no protein level change. B. Summary of alanine-scanning results. The first 40 residues of E are shown. The TM domain and the secondary structure are indicated above. Colors of each residue are according to A. C. A model for the first α-helix. Colors are according to A. Residues from growth curves in A are labeled. Asterisks indicate residues changed to phenylalanine later in the text.

Fig. 3

Effect of leucine mutations. A. Sequences of the wild-type, leucine variants and the L16P/P21L mutant. Underlines indicate positions of residues that vary in the leucine variants. Asterisks indicate the position of mutated residues in the L16P/P21L mutant. B. Growth curves of the mutants in TOP10 cells. C. (left) Western blot of whole cells for the leucine mutants. An unknown secondary protein product is evident in E-L_N. (right) Western blot of membrane fractions.

Fig. 4

Defining the minimal length of E required for lysis activity. A. Illustration of the lysozyme fusion constructs. e.g. E₂₅ contains the first 25 residues followed by the linker and an inactive lysozyme shown after the arrow. B. Growth curves of wild-type and the E_25–28 fusion constructs in TOP10 cells. Inset, Western blot of whole cells after 10 min of induction. C. Illustration of the N-terminal deletion constructs. D. Growth curves of deletion constructs in TOP10 cells. Inset, Western blot of whole cells after 10 min of induction.

Fig. 5

Lysis activity of the minimal region. A. Illustration of the minimal region constructs that contains residues 11 to 28 of E followed by the linker and an inactive lysozyme. The asterisk indicates the position of Leu19. B. Growth curves of wild-type and various minimal region constructs in TOP10 cells. Insert, Western blot of both whole cells and the membrane fraction after 10 min of induction. C. Growth curves of all Leu-to-Phe mutants of the minimal region in TOP10 cells. Inset, Western blot of the membrane fraction after 10 min of induction. L13F failed to insert into the membrane.

Fig. 6

Protein E forms a stable complex with MraY. Tagged wild-type E and E variants were expressed and purified on the Ni-NTA column along with a control (an empty pRSFDuet vector). The presence of the E variants and captured native MraY were confirmed by western blot using anti-penta-His and anti-MraY. Molecular weights of wild-type, P21A, E-LS, E-L_N, and E-L_C are comparable, but all variants have different mobility on the gel. Similarly, E_{11–28(L19F)} runs slower than E₂₈ on the gel despite its smaller molecular weight likely differences in charge. Two bands of native MraY are visible under these conditions.