In Vivo Bypass of Chaperone by Extended Coiled-Coil Motif in T4 Tail Fiber

Abstract

The distal-half tail fiber of bacteriophage T4 is made of three gene products: trimeric gp36 and gp37 and monomeric gp35. Chaperone P38 is normally required for folding gp37 peptides into a P37 trimer; however, a temperature-sensitive mutation in T4 (ts3813) that suppresses this requirement at 30°C but not at 42°C was found in gene 37 (R. J. Bishop and W. B. Wood, Virology 72:244-254, 1976). Sequencing of the temperaturesensitive mutant revealed a 21-bp duplication of wild-type gene 37 inserted into its C-terminal portion (S. Hashemolhosseini et al., J. Mol. Biol. 241:524-533, 1994). We noticed that the 21-amino-acid segment encompassing this duplication in the ts3813 mutant has a sequence typical of a coiled coil and hypothesized that its extension would relieve the temperature sensitivity of the ts3813 mutation. To test our hypothesis, we crossed the T4 ts3813 mutant with a plasmid encoding an engineered pentaheptad coiled coil. Each of the six mutants that we examined retained two amber mutations in gene 38 and had a different coiled-coil sequence varying from three to five heptads. While the sequences varied, all maintained the heptad-repeating coiled-coil motif and produced plaques at up to 50°C. This finding strongly suggests that the coiled-coil motif is a critical factor in the folding of gp37. The presence of a terminal coiled-coil-like sequence in the tail fiber genes of 17 additional T-even phages implies the conservation of this mechanism. The increased melting temperature should be useful for "clamps" to initiate the folding of trimeric ␤-helices in vitro and as an in vivo screen to identify, sequence, and characterize trimeric coiled coils.Escherichia coli bacteriophage T4 is a complex nanomachine assembled from DNA and protein. It is composed of four major structural components: a genome of DNA, a head, a tail, and tail fibers (TFs) (Fig. 1). Most, or all, T4 virion structural proteins are transcribed and translated during the late period of infection. The assembly processes of the three protein structural components (head, tail, and TFs) occur in parallel with the aid of a group of chaperones (39,40). Nearly all of these chaperones are proteins encoded by genes in the T4 genome (10). They help to regulate the folding and assembly of T4 proteins and are not included in the final functional structure of the virion.The T4 long TFs (LTFs) are critical for the first steps of viral infection (11,12). The chemosensors in the carboxy-terminal region of the LTFs recognize the host cell by binding (reversibly) to receptors displayed on the cell surface (14). The phage then attaches to the cell irreversibly via P12 (short TFs) (31). Each LTF on the phage has a rod-like structure that consists of proximal-and distal-half TFs of approximately equal lengths, joined at their ends in an angle (39). Proximal-half LTFs are parallel homotrimers of matured gp34 (called P34, where P denotes the homooligomeric assembly of the monomeric gene product, gp34). Distal-half fibers contain a monomer of gp35, a homotrimer of ...

Bacteriophage T4 long tail fiber domains

“…To test this possibility, Goldberg and colleagues (including one of this review's authors, PH) constructed phage containing even longer duplications of the putative coiled-coil α-helix (Qu et al 2004). These motifs consist of a minimal amino acid heptad which forms two turns of an α-helix.…”

Lean forward: Genetic analysis of temperature‐sensitive mutants unfolds the secrets of oligomeric protein complex assembly

“…Sequencing revealed that the mutant gp37 had a duplication of a sequence in its C terminus . Recognizing that the duplicated sequence included hydrophobic heptad repeats, Qu et al introduced artificial coiled‐coil‐forming sequences into wild‐type gp37 and found that this was sufficient to bypass the need for the P38 chaperone . Moreover, multiple coiled‐coil‐forming insertions allowed phage function at extremely high temperatures .…”

“…Recognizing that the duplicated sequence included hydrophobic heptad repeats, Qu et al introduced artificial coiled‐coil‐forming sequences into wild‐type gp37 and found that this was sufficient to bypass the need for the P38 chaperone . Moreover, multiple coiled‐coil‐forming insertions allowed phage function at extremely high temperatures . By promoting early homomeric gp37 associations even at temperatures that destabilized wild‐type gp37 assembly/folding intermediates, the extra‐coiled coils were sufficient to direct the proper assembly of the tail fiber without assistance from P38.…”

Characterization of Extended-Host-Range Pseudo-T-Even Bacteriophage Kpp95 Isolated on Klebsiella pneumoniae

“…However, the mechanisms involved in these phages' abilities to infect different hosts have not been elucidated. To determine the sequence which might be involved in Kpp95 adsorption, we tried to obtain Kpp95 gene 37 by PCR amplification with primers specific to T4 gene 37 (37RV-1F [5Ј-GTTCTGGTAATTTTGCTAAC-3Ј] and 37RV-1R [5Ј-A ACAGCTAACTTTGGATATG-3Ј], FR86 [5Ј-GCTTCAAGT ACTGACTTAGG-3Ј], and FR89 [5Ј-ACAGTGATAGTATG ACCATGTGATCC-3Ј]) (37,49). However, the reactions failed to give a PCR product.…”