In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 117, No. 47 ( 2020-11-24), p. 29637-29646
Abstract:
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).
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.2007979117
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2020
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
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