An elongated spine of buried core residues necessary for in vivo folding of the parallel beta-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.

Fig. 1.

Side chain stacking in the core of the P22 tailspike β-helix. (A) The C-terminal portion (113–666) of a single chain of the P22 tailspike trimer is shown as a ribbon diagram and a transparent molecular surface. Backbone and side chain atoms of inward pointing, buried core residues are shown in space-fill and colored according to their stack. Solvent-exposed residues are not shown for clarity. (B) Rung 6 cross-section of the β-helix domain, demonstrating the locations of inward-pointing stacks (spheres) and outward-pointing residues (sticks). (C) Side chain atoms of the buried core. Single spheres represent glycines. (Left) The same orientation as A. (Right) Rotated by 180°.

Fig. 2.

In vivo folding pathway of the P22 tailspike homotrimer, as described in the text. The left branch of the folding pathway shows productive intracellular intermediates leading to the native state. Intermediates leading to the inclusion body state are on the right. [pT], protrimer.

Fig. 3.

Sample SDS/PAGE data of complete cellular lysates. (A) Positive (WT and ΔN) and negative (pET11a) control samples expressed at 37°C. WT and ΔN expression samples produce some native, SDS-resistant trimers (NT) as well as nonnative, SDS-sensitive chains that unfold (U) upon mixture with SDS. WT and mutant samples are shown for expression at 37°C (B), 30°C (C), and 18°C (D).

Fig. 4.

Quantitative results of folding experiments. Average percentages of WT folding efficiencies are shown for all alanine mutants when expressed at 37°C (red circles), 30°C (brown triangles), and 18°C (blue squares). Colored lines indicate the standard deviations of triplicate experiments. Note that WT and ΔN average percentage of WT folding efficiencies and standard deviations are shown as Insets. The 75% threshold delineating WT-like folders and folding-deficient samples is shown as a dashed line.

Fig. 5.

Folding efficiencies mapped onto the native structure of the tailspike β-helix. Mutated positions are colored according to the folding efficiency of the residue’s alanine mutant. Positions that are glycine in the WT sequence or fold like WT are shown by stick representation. Folding-deficient positions are shown as a surface volume.

Fig. 6.

Thermostability assay on 18°C WT-like folders. The percentage of native-tailspike chains completely unfolded in the presence of 2% SDS when heated to 80°C (blue circle) or 90°C (red square) is shown as the average of duplicate experiments. Lines represent standard deviations. WT and ΔN controls are shown in the Inset.

Fig. 7.

Model of the processive folding of the β-helix. Critical internal side chain contacts (cyan ovals) nucleate an initial set of well formed rungs. This nucleus acts as a template for other rungs to assemble by means of side chain stacking and/or tethering around loop regions. Capping of the N terminus likely prevents aggregation.