Structural and functional characterization of the pore-forming domain of pinholin S

Pinholin S(21)68 triggers the lytic cycle of bacteriophage φ21 in infected Escherichia coli. Activated transmembrane dimers oligomerize into small holes and uncouple the proton gradient. Transmembrane domain 1 (TMD1) regulates this activity, while TMD2 is postulated to form the actual “pinholes.” Focusing on the TMD2 fragment, we used synchrotron radiation-based circular dichroism to confirm its α-helical conformation and transmembrane alignment. Solid-state (15)N-NMR in oriented DMPC bilayers yielded a helix tilt angle of τ = 14°, a high order parameter (S(mol) = 0.9), and revealed the azimuthal angle. The resulting rotational orientation places an extended glycine zipper motif (G(40)xxxS(44)xxxG(48)) together with a patch of H-bonding residues (T(51), T(54), N(55)) sideways along TMD2, available for helix–helix interactions. Using fluorescence vesicle leakage assays, we demonstrate that TMD2 forms stable holes with an estimated diameter of 2 nm, as long as the glycine zipper motif remains intact. Based on our experimental data, we suggest structural models for the oligomeric pinhole (right-handed heptameric TMD2 bundle), for the active dimer (right-handed Gly-zipped TMD2/TMD2 dimer), and for the full-length pinholin protein before being triggered (Gly-zipped TMD2/TMD1-TMD1/TMD2 dimer in a line).