Fibritin Encoded by Bacteriophage T4 Gene wac has a Parallel Triple-stranded α-Helical Coiled-coil Structure

The bacteriophage T4 late gene wac (whiskers antigen c ontrol) 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% α-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 residue, 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 α-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 α-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 α-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 α-fibrous proteins.