Inhibition of Escherichia coli ATP synthase by amphibian antimicrobial peptides

Abstract

Previously melittin, the alpha-helical basic honey bee venom peptide, was shown to inhibit F(1)-ATPase by binding at the beta-subunit DELSEED motif of F(1)F(o)-ATP synthase. Herein, we present the inhibitory effects of the basic alpha-helical amphibian antimicrobial peptides, ascaphin-8, aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, citropin 1.1, dermaseptin, maculatin 1.1, maganin II, MRP, or XT-7, on purified F(1) and membrane bound F(1)F(0)Escherichia coli ATP synthase. We found that the extent of inhibition by amphibian peptides is variable. Whereas MRP-amide inhibited ATPase essentially completely (approximately 96% inhibition), carein 1.8 did not inhibit at all (0% inhibition). Inhibition by other peptides was partial with a range of approximately 13-70%. MRP-amide was also the most potent inhibitor on molar scale (IC(50) approximately 3.25 microM). Presence of an amide group at the c-terminal of peptides was found to be critical in exerting potent inhibition of ATP synthase ( approximately 20-40% additional inhibition). Inhibition was fully reversible and found to be identical in both F(1)F(0) membrane preparations as well as in isolated purified F(1). Interestingly, growth of E. coli was abrogated in the presence of ascaphin-8, aurein 2.2, aurein 2.3, citropin 1.1, dermaseptin, magainin II-amide, MRP, MRP-amide, melittin, or melittin-amide but was unaffected in the presence of carein 1.8, carein 1.9, maculatin 1.1, magainin II, or XT-7. Hence inhibition of F(1)-ATPase and E. coli cell growth by amphibian antimicrobial peptides suggests that their antimicrobial/anticancer properties are in part linked to their actions on ATP synthase.

Figure 1. X-ray structures (A) of mitochondrial ATP synthase showing βDELSEED residues and (B)α-helical structure of melittin and magainin II

Rasmol software was used to generate these figures. For ATP synthase β-subunit DELSEED PDB file 1H8E was used [49]. βDELSEED is the proposed peptide binding site E. coli residue numbering is shown. The 26-residue long structure of the α-helical honey bee (Apis mellifera) venom peptide melittin, was generated by using PDB file 2MLT [50]. Five positively charged residues along with amide group (NH₂) on the c-terminal are identified. The 23-residue long structure of the α-helical amphibian (Xenopus laevis) peptide magainin II, was generated by using PDB file 2MAG [51]. Three positively charged residues along with amide group (NH₂) on the c-terminal are identified.

Figure 2. Inhibition of ATPase activity in purified F₁ or membrane-bound ATP synthase by melittin, melittin-amide, MRP, MRP-amide

Membranes or purified F₁ were preincubated for 60 min at 23°C with varied concentration of melittin, melittin-amide, MRP, MRP-amide, and then 1 ml of assay buffer was added and ATPase activity determined. Details are given in Materials and Methods. Symbols used are: circles (○, melittin –amide and Δ, melittin), triangles (○, MRP-amide and Δ, MRP). Each data point represents average of at least four experiments done in duplicate tubes, using two independent membrane or F₁ preparations. Results agreed within ± 10%.

Figure 3. Inhibition of ATPase activity in purified F₁ or membrane-bound ATP synthase by aurein 2.2, aurein 2.3, carein 1.8, carein 1.9, magaininII, or magainin II-amide

Purified F₁ or membranes were preincubated for 60 min at 23°C with varied concentration of aurein 2.2 (○), aurein 2.3 (Δ), carein 1.8 (Δ), carein 1.9 (○), magaininII (Δ), or magainin II-amide (○) and then 1 ml of assay buffer was added and ATPase activity determined. Details are given in Materials and Methods. Each data point represents average of at least four experiments done in duplicate tubes, using two independent membrane or F₁ preparations. Results agreed within ± 10%.

Figure 4. Inhibition of ATPase activity in purified F₁ or membrane-bound ATP synthase by ascaphin-8, citropin 1.1, dermaseptin, maculatin 1.1, or XT-7

Purified F₁ or membranes were preincubated for 60 min at 23°C with varied concentration of ascaphin-8, citropin 1.1, dermaseptin, maculatin 1.1, or XT-7 and then 1ml of assay buffer was added and ATPase activity determined. Details are given in Materials and Methods. Each data point represents average of at least four experiments done in duplicate tubes, using two independent membrane or F₁ preparations. Results agreed within ± 10%.

Figure 5. Results of Extra pulse of peptides on purified F₁ or membrane bound ATP synthase

Membrane bound ATP synthase (Mbr) or purified F₁ (F₁) was inhibited with inhibitory concentrations of amphibian peptides or melittin for 60 min under conditions as described in Fig 2–4. Then a further pulse of identical inhibitory concentrations was added and incubation continued for 1 h before assay. The last digits represent the peptide concentrations in [µM].

Figure 6

Results of reversal of inhibition by passing through centrifuge columns purified F₁ (F₁) was inhibited with inhibitory concentrations of the peptides shown in the figure for 60 min under conditions as described in Fig 2–4. Then the inhibited samples were passed twice through 1 ml centrifuge columns and ATPase activity was measured. Identical reversibility results were obtained by diluting the inhibited membrane bound enzyme or purified F₁. For clarity, only the data for reversibility by centrifuge columns is shown.