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  • 标题:Structural and functional characterization of the pore-forming domain of pinholin S2168
  • 本地全文:下载
  • 作者:Lena M. E. Steger ; Annika Kohlmeyer ; Parvesh Wadhwani
  • 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
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