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

Abstract

The parallel beta-helix is an elongated beta-sheet protein domain associated with microbial virulence factors, toxins, viral adhesins, and allergens. Long stacks of similar, buried residues are a prominent feature of this fold, as well as the polypeptide chain fold of an amyloid structure. The 13-rung, right-handed, parallel beta-helix of the homotrimeric P22 tailspike adhesin exhibits predominantly hydrophobic stacks. The role of these stacked residues in the folding and stabilization of the protein is unclear. Through scanning alanine mutagenesis we have identified a folding spine of stacked residues in continuous contact along the length of P22 tailspike's beta-helix domain that is necessary for folding within cells. Nearly all chains carrying alanine substitutions of the 103 buried nonalanines were defective in folding in vivo at 37 degrees C. However, the majority of these chains successfully reached a native state, stable to >80 degrees C, when folded inside cells at low temperatures. Thus, nearly the entire buried core was critical for in vivo beta-helix folding but negligible for stability. Folding at 18 degrees C revealed the minimal folding spine of 29 nonglycine stack positions that were intolerant to alanine substitution. These results indicate that a processive folding mechanism, dependent on stacking contacts, controls beta-helix formation. Such a stepwise folding pathway offers a new target for drug design against this class of microbial virulence factors.