 |
|
Title
|
|
Fibritin encoded by bacteriophage T4 gene wac has a parallel triple-stranded alpha-helical coiled-coil structure.
|
|
|
Authors
|
|
V.P.Efimov,
I.V.Nepluev,
B.N.Sobolev,
T.G.Zurabishvili,
T.Schulthess,
A.Lustig,
J.Engel,
M.Haener,
U.Aebi,
Venyaminov SYu.
|
|
|
Ref.
|
|
J Mol Biol, 1994,
242,
470-486.
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
The bacteriophage T4 late gene wac (whisker's antigen control) encodes a fibrous
protein which forms a collar/whiskers complex. Whiskers function as a helper
protein for the long tail fibres assembly and plays a role in regulating
retraction of the long tail fibres in response to environmental conditions. In
this work we show that expression of the cloned wac gene in Escherichia coli
yields a protein oligomer of 53 nm length which we call fibritin, and which is
able to complement gpwac T4 particles in vitro. CD spectroscopy of fibritin
indicates a 90% alpha-helical content, and scanning calorimetry shows that the
protein has several distinct domains. The analysis of the 486 amino acid
sequence of fibritin reveals three structural components: a 408 amino acid
region that contains 12 putative coiled-coil segments with a canonical heptad
(a-b-c-d-e-f-g)n substructure where the "a" and "d" positions are preferentially
occupied by apolar residues, and the N and C-terminal domains (47 and 29 amino
acid residues, respectively) have no heptad substructure. The distribution of
hydrophobic residues within heptads is more similar to a triple than to a double
coiled-coil. The alpha-helical segments are separated by short "linker" regions,
variable in length, that have a high proportion of glycine and proline residues.
Each coiled-coil segment has, on the borders with linker regions, residues that
are common to the N and C-terminal caps of the alpha-helices. Full-length and
amino-terminally truncated fibritins can be reassembled in vitro after
temperature-induced denaturation. Co-assembly of full-length fibritin and the
N-terminal deletion mutant, as well as analytical centrifugation, indicates that
the protein is a parallel triple-standard alpha-helical coiled-coil. Deletions
of various N-terminal portions of fibritin did not block trimerisation but the
mutant trimers are unable to bind to T4 particles. The last 18 C-terminal
residues of fibritin are required for correct trimerisation of gpwac monomers in
vivo. We propose that fibritin might serve as a convenient model for the
investigation of folding and assembly mechanisms of alpha-fibrous proteins.
|
|
|
|