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

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. ​(Fig.1).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. FIG. 1. Bacteriophage T4 structures. (A) Virion; (B) TF self-assembly. 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 gp36 (P36), and a homotrimer of gp37 (P37). Though it had long been suspected that P34, P36, and P37 were dimeric in mature LTFs with only a single copy of gp35 (5, 7), the Steven lab (4) demonstrated by scanning transmission electron microscopy that P34, P36, and P37 were trimeric homooligomers. We have confirmed this finding by analytical ultracentrifugation and comparison of the molecular weights of whole LTFs to LTFs with 346 (out of 1,026) residues deleted from gp37 (Y. Sheik, W. Stafford, P. Hyman, and E. B. Goldberg, unpublished data). The folding and oligomerization of the TFs is regulated by chaperones encoded by genes 57A (23) and 38 (9). Chaperone P57A participates in the assembly of P34 and P37 and also in the assembly of the short TF, P12 (15, 19). P38 is necessary (in addition to P57A) for the trimerization of gp37 to form the mature P37 rod, which initiates the extension of LTF assembly (9, 13). The order of assembly of the distal-half fiber starts with the maturation of three gp37 monomers into the P37 trimer. Then, three gp36 monomers bind to the N terminus of the P37 trimer and mature into the P36-P37 rod. This event is followed by the addition of one gp35 molecule to the N terminus of the mature trimeric P36 LTF segment to complete the distal-half fiber. The P34 trimer can then join to the gp35 end of the distal-half fiber at an angle to form a complete LTF (20). Although it is certain that chaperones P57A and P38 are involved in distal-half fiber assembly, the binding sites, order of interactions, and mechanisms of their functions are still not clear. A temperature-sensitive mutation (ts3813) that suppresses the requirement for P38 function at 30°C but not at 42°C was found in gene 37 (which codes for gp37) (2). The authors initially speculated that P38 may catalyze the noncovalent association of two gp37 molecules. A comparison of the wild-type T4 sequence of gene 37 to the mutant sequence (12) showed a 21-bp duplication of gene 37 inserted between nucleotides 3328 and 3329, near its C terminus (Table ​(Table1,1, first and second rows). They speculated that the extended sequence is responsible for the maturation of gp37 to P37 in the absence of P38. We recognized that the extended 21-nucleotide sequence in the ts3813 mutant had a coiled-coil (CC) motif within a CC diheptad present in wild-type T4. When we tested the peptide sequence by using COILS (http://www.ch.embnet.org/software/COILS_form.html), the extended 21-nucleotide sequence in the ts3813 mutant was predicted to have a 98.4% CC-forming probability (when the window size was 21 residues), while in wild-type gene 37 protein, the same region of a 15-amino-acid (aa) sequence exhibited much lower CC formation probabilities (0.714 with a window size of 14 residues and 0.020 with a window size of 21 residues). Cerritelli et al. also examined the protein sequence of all of the segments of wild-type T4 TF (4) and, although 2.5 “possible” heptads were identified in this region, concluded that α-helical coiled coils were “at most a minimal element in the LTF.” TABLE 1. DNA and amino acid sequences of recombinant T4 phages We hypothesized that a short trimeric CC that was brought into alignment and stabilized by the P38 chaperone protein could efficiently initiate the upstream folding of the thick β-structural rod (1, 4, 17). The corollary was that by adding the third heptad, the enhanced stability of the ts3813 bypass mutation would make an intrinsically more stable CC (a clamp) and thereby relieve the requirement for P38 at 30°C but not at 42°C (2). (We found that the ts3813 mutation fails to suppress P38 mutations even at 37°C.) In this paper, we report the design and construction of a series of T4 phages with longer or putatively more stable CC-like heptads in gp37 that enable P38 chaperone bypass at temperatures ranging from 30 to 50°C in vivo. From the phenotypic differences of the T4 series and the fact that all of the bypass mutants that we found had 7-, 14-, or 21-residue inserts, we conclude that the extended CC region is responsible for the initiation of P37 trimeric assembly and acts as an artificial cis chaperone to bypass the need for P38.