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标题：Structural and functional characterization of the pore-forming domain of pinholin S2168
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作者：Lena M. E. Steger ; Annika Kohlmeyer ; Parvesh Wadhwani 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2020
卷号：117
期号：47
页码：29637-29646
DOI：10.1073/pnas.2007979117
出版社：The National Academy of Sciences of the United States of America
摘要：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).
关键词：pinholin ; transmembrane protein ; glycine zipper ; solid-state NMR ; synchrotron circular dichroism