Buried hydrophobic side‐chains essential for the folding of the parallel β‐helix domains of the P22 tailspike

Abstract

The processive ␤-strands and turns of a polypeptide parallel ␤-helix represent one of the topologically simplest ␤-sheet folds. The three subunits of the tailspike adhesin of phage P22 each contain 13 rungs of a parallel ␤-helix followed by an interdigitated section of triple-stranded ␤-helix. Long stacks of hydrophobic residues dominate the elongated buried core of these two ␤-helix domains and extend into the core of the contiguous triple ␤-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 ␤-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 ␤-helix and contiguous ␤-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 ␤-helix, triple-stranded ␤-helix, and ␤-prism domains in the tailspike protein.

An elongated spine of buried core residues necessary for in vivo folding of the parallel β-helix of P22 tailspike adhesin

“…In contrast to the insignificant effect that removal of Ͼ100 aa had on the folding of the ⌬N tailspike protein, alanine substitution of any one of 95 of the 103 buried core sites significantly hindered the tailspike chains' ability to reach the native state when expressed at 37°C. One of these mutants, F308A, was previously shown to fail to fold at high and low temperatures (22). Our high-throughput system was able to accurately reproduce the published data for this residue as well as all other ␤-helix residues investigated by Betts et al (22).…”

“…One of these mutants, F308A, was previously shown to fail to fold at high and low temperatures (22). Our high-throughput system was able to accurately reproduce the published data for this residue as well as all other ␤-helix residues investigated by Betts et al (22). Of the remaining 42 predominantly surface-exposed sites, 20 had defective folding phenotypes.…”

“…This finding supports the notion that side chain stacks are necessary to encode an elongated ␤-helix. This result is in contrast to the large number of previously isolated tsf mutations that represent solventexposed residues critical in folding at high temperatures but unnecessary at lower temperatures (22). The short core stacks may function to stabilize intermediate forms or to specifically prevent off-pathway aggregation.…”

“…In contrast, five phenylalanines and one leucine in the hydrophobic core stacks are essential for the folding, but not stability, of tailspike at high and low temperatures (22). This finding suggests that stacked core residues contain the necessary sequence information to direct ␤-helix formation.…”

“…Upon release from the ribosome tailspike, thermolabile monomeric intermediates, [I], may oligomerize and further fold into the SDS-resistant trimeric tailspike, N (21). Mutations that block oligomerization or trimer maturation result in the accumulation of soluble, SDS-sensitive species (18,22). Alternatively, the presence of any of Ͼ60 genetically isolated temperature-sensitive folding (tsf ) mutations at temperatures in the higher range of cellular growth will generate a misfolded monomeric conformation, [I*], which specifically self-aggregates and accumulates in inclusion bodies (23).…”