Buried hydrophobic side-chains essential for the folding of the parallel beta-helix domains of the P22 tailspike

Abstract

The processive beta-strands and turns of a polypeptide parallel beta-helix represent one of the topologically simplest beta-sheet folds. The three subunits of the tailspike adhesin of phage P22 each contain 13 rungs of a parallel beta-helix followed by an interdigitated section of triple-stranded beta-helix. Long stacks of hydrophobic residues dominate the elongated buried core of these two beta-helix domains and extend into the core of the contiguous triple beta-prism domain. To test whether these side-chain stacks represent essential residues for driving the chain into the correct fold, each of three stacked phenylalanine residues within the buried core were substituted with less bulky amino acids. The mutant chains with alanine in place of phenylalanine were defective in intracellular folding. The chains accumulated exclusively in the aggregated inclusion body state regardless of temperature of folding. These severe folding defects indicate that the stacked phenylalanine residues are essential for correct parallel beta-helix folding. Replacement of the same phenylalanine residues with valine or leucine also impaired folding in vivo, but with less severity. Mutants were also constructed in a second buried stack that extends into the intertwined triple-stranded beta-helix and contiguous beta-prism regions of the protein. These mutants exhibited severe defects in later stages of chain folding or assembly, accumulating as misfolded but soluble multimeric species. The results indicate that the formation of the buried hydrophobic stacks is critical for the correct folding of the parallel beta-helix, triple-stranded beta-helix, and beta-prism domains in the tailspike protein.

Figure 1.

Features of the P22 tailspike protein. (A) Ribbon diagram of the P22 tailspike protein (Steinbacher et al. 1994, 1996). The trimer is oriented with the amino termini up and the carboxyl termini down. The subunits are in different colors. The single parallel β-helix domain (residues 143–540) is below the amino-terminal capsid-binding domain (Steinbacher et al. 1996). The narrower bottom portion of the structure includes the triple-stranded β-helix (residues 541–558), an intertwined region in which the three independent strands wrap around each other. Following this region, the chains fold back on themselves for 5 rungs—but still with a common buried core—called the triple β-prism (residues 559–619) and then twist into the cysteine annulus. They then separate to form the β-sheet caudal fins. Residues targeted by mutagenesis are labeled; these and other residues that form the triple β-prism subunit interface are shown in space-filling mode. (B) Depiction of the single parallel β-helix domain showing two of the three interior side-chain stacks, stack A (cyan) and stack C (green); the backbone atoms are shown using CPK color scheme residues mutated in this paper are indicated. (C) Axial views of three stacked coils that form the triple-stranded β-helix and the triple β-prism domains. Isoleucine and leucine side-chains from each subunit can be seen at the center of each coil forming the hydrophobic core of the interdigitated domain. Sequences in connecting loops and turns are omitted. Purple stars identify Ile 548 (top) and Ile 560 (middle). Yellow stars mark Glu 543.

Figure 2.

Tailspike folding and aggregation pathways, in vivo and in vitro. Newly synthesized chains released from ribosomes form a single chain partially folded intermediate [I] (Clark and King 2001). For chains refolded out of denaturant an earlier intermediate [I_o] is suggested from spectroscopy (Fuchs et al. 1991). The [I] intermediate folds further to a species that can self-associate to form the protrimer intermediate [pT], in which the chains are associated but not fully folded (Goldenberg and King 1982; Fuchs et al. 1991; Betts and King 1999). The protrimer, a disulfide bonded species, then transforms to the native tailspike [Nt]. Interchain disulfide bonds stabilize the protrimer, but are reduced in the transition to the native trimer (Robinson and King 1997). The early single chain species [I] is thermolabile, and as temperature increases, forms a species [I^*] that associates into multimers that polymerize into the kinetically trapped inclusion body state.

Figure 3.

Structure-based sequence alignment of the parallel β-helix domain. The figure is a simplified alignment to guide the reader. The first column indicates the rung number (1–14), and the second column indicates the position of the first residue in each rung. The column labels indicate if the side-chains point to the inside (i) or the outside (o) of the β-helix. Colors identify residues in the three β-sheets (cyan, sheet A; yellow, sheet B; green, sheet C). The boxes identify the seven residues targeted by site-directed mutagenesis as well as the two stacks shown in corresponding colors in Figure 1B ▶. Residues that are the sites of tsf mutants are shown in red, i.e., tsfT307A and tsfE309V, and the locations of the two global suppressors of tsf mutations (V331A and A334V) are in blue.

Figure 4.

Substitutions of stacked phenylalanine residues in parallel β-sheet C; SDS gel electrophoresis of the soluble and aggregated chains. Tailspike expression in E. coli was induced by addition of IPTG, and cells were harvested after 2 h expression at 30°C. Supernatant and pellet fractions of cell lysates expressing single amino acid replacements at tailspike residues Phe 284 (A), Phe 308 (B), and Phe 336 (C) were electrophoresed through SDS-polyacrylamide gels. The gels were stained with Coomassie blue. The small amount of native tailspike in the pellet is due to incomplete lysis of the cells.

Figure 5.

In vivo partitioning of mutant tailspike chains between productive folding and aggregated inclusion bodies. The levels of tailspike chains were determined by laser densitometry as described in Materials and Methods. The data are plotted as averages of duplicate cultures expressing tailspike chains that contained single amino acid replacements at residues Phe 181 (A), Phe 284 (B), Phe 308 (C), and Phe 336 (D). Cultures expressing wild-type tailspike were included in each experiment as controls. The SDS-polypeptide chain complexes found in the pellet represent chains solubilized from the inclusion body state.

Figure 6.

Effect of temperature on in vivo folding of tailspike chains. Cultures expressing wild-type, F308A, and F308V tailspike chains were induced with IPTG and portions were immediately transferred to different temperatures for continued incubation (18°C for 5 h, 30°C for 3.5 h; and 37° and 39°C for 3 h). Cultures were harvested, lysed, and separated into supernatant and pellet fractions. The wild-type value, indicated by the solid gray bar, is defined as 100%. F308A strain, black bar with white spots; F308V strain, white bar with black spots.

Figure 7.

In vivo partitioning of tailspike chains containing substitutions in a single stack in parallel β-sheet A. Cultures of Leu 144 (top), Phe 381 (middle), and Phe 540 (bottom) were fractionated into low-speed pellet and supernatant fractions, and the polypeptide chains electrophoresed through SDS-polyacrylamide gels. The majority of the chains recovered from the supernatant migrated similarly to SDS-resistant native-like trimers. The chains in the pellet migrated as SDS-soluble inclusion body chains. The positions of the targeted substitutions are indicated in Figure 1B ▶ and the alignment is Figure 3 ▶ (boxed residues). The levels of tailspike chains were determined by laser densitometry as described in Materials and Methods. The data are plotted as averages of duplicate cultures expressing tailspike chains. Cultures expressing wild-type tailspike were included in each experiment as controls.

Figure 8.

In vivo partitioning of tailspike chains containing replacements of the buried side-chain residues of the triple-stranded β-helix and triple β-prism domains. (A) Cultures expressing vector, wild-type, E543I, I548V, I548A, I560V, I560A, and F284I chains were fractionated into low-speed pellet and supernatants. Samples were electrophoresed through SDS-polyacrylamide gels and stained with Coomassie blue. (B) Quantification of the tailspike bands in the above gel by laser densitometry. (C) Examination of the conformational properties of tailspike species recovered from supernatant fractions, as analyzed by native gel electrophoresis in the cold. The slightly retarded mobility of E543I is expected for a substitution that changes the net surface charge on the tailspike (Yu and King 1988). The slower mobilities of the I548A chains are associated with the protrimer intermediate (Benton et al. 2002) or multimeric aggregation intermediates (Speed et al. 1996).