Abstract
Gene product 5 (gp5) of bacteriophage T4 is a spike-shaped protein that functions to disrupt the membrane of the target cell during phage infection. Its C-terminal domain is a long and slender β-helix that is formed by three polypeptide chains wrapped around a common symmetry axis akin to three interdigitated corkscrews. The folding and biophysical properties of such triple-stranded β-helices, which are topologically related to amyloid fibers, represent an unsolved biophysical problem. Here, we report structural and biophysical characterization of T4 gp5 β-helix and its truncated mutants of different lengths. A soluble fragment that forms a dimer of trimers and that could comprise a minimal self-folding unit has been identified. Surprisingly, the hydrophobic core of the β-helix is small. It is located near the C-terminal end of the β-helix and contains a centrally positioned and hydrated magnesium ion. A large part of the β-helix interior comprises a large elongated cavity that binds palmitic, stearic, and oleic acids in an extended conformation suggesting that these molecules might participate in the folding of the complete β-helix.
Highlights
Bacteriophage T4 uses a large multicomponent organelle, called a tail, for recognition and attachment to an Escherichia coli cell [1,2]
The recombinant proteins were expressed as fusion constructs at 37 ̋ C in the E. coli BL21 (DE3) host strain (Novagen, Darmstadt, Germany) after IPTG induction to a final concentration of 1 mM
More than 78% of the complete gp5 β-helical domain—18 out of 23 β-strands (Figure 1)—is fully with each eachamino aminoacid acidin in a given polypeptide chain interacting two amino interdigitated with a given polypeptide chain interacting withwith two amino acids acids from fromsame the chain same chain each β-strand) andtofour to six amino acidstwo from twopolypeptide other polypeptide the (within(within each β-strand) and four six amino acids from other chains chains forming the
Summary
Bacteriophage T4 uses a large multicomponent organelle, called a tail, for recognition and attachment to an Escherichia coli cell [1,2]. Gene product (gp) 5 is critical for the assembly of the tail’s baseplate [3,4,5] and for the tail function during infection [6]. A spike-shaped trimeric protein, gp forms the centerpiece of the baseplate [7]. It has long been hypothesized to function as a piercing needle with which the phage disrupts the multilayered host cell envelope upon attachment to the cell surface and subsequent sheath contraction [7]. The amino acid sequence of gp contains 575 residues that form three domains connected by long linkers. Residues 1–129, 174–339, and 389–575 comprise the N-terminal
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