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

Abstract

The parallel ␤-helix is an elongated ␤-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 ␤-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 ␤-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°C. However, the majority of these chains successfully reached a native state, stable to >80°C, when folded inside cells at low temperatures. Thus, nearly the entire buried core was critical for in vivo ␤-helix folding but negligible for stability. Folding at 18°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 ␤-helix formation. Such a stepwise folding pathway offers a new target for drug design against this class of microbial virulence factors.buried stacks ͉ protein folding ͉ folding mutants

Structural and Functional Insights into Intramolecular Fructosyl Transfer by Inulin Fructotransferase

“…The side chains of adjacent subunits interdigitate, which stabilizes the trimer (38,39). The surface area buried by trimerization is calculated to be 4439 Å 2 /monomer, or 28% of the monomer surface area.…”

“…Tailspike protein, which forms a trimer in a manner similar to that of BsIFTase, is the exception, although its proposed catalytic mechanism occurs within a monomeric structural context (31). Therefore, trimerization of the tailspike protein ␤-helix may not be required for catalysis and might instead be important for thermostability and protease resistance (38,39). In contrast to that of tailspike protein, the BsIFTase active site is located at the monomer-monomer interface.…”

A Conserved Aromatic Residue in the Autochaperone Domain of the Autotransporter Hbp Is Critical for Initiation of Outer Membrane Translocation

“…The importance of residues that are buried in the hydrophobic core in processive folding seems to be a general feature of ␤-helical segments. In the P22 tailspike protein, bulky hydrophobic side chains appear to be essential for folding (17).…”

“…A ␤-helix is an extremely stable fold of ␤-strands, in this case of triangular rungs comprising three strands, that are interrupted by loops with variable length and are stacked to form a cross ␤-structure (15,16). Other examples of this repetitive fold include P22 tailspike protein, pectate lyase, and a domain of the HET-s prion protein (17)(18)(19). Importantly, most of the hundreds of ATs analyzed are predicted to include extensive ␤-helical structure, suggesting a generic role in biogenesis and transport (20,21).…”

Dissociation of intermolecular disulfide bonds in P22 tailspike protein intermediates in the presence of SDS

“…Here we show that when the oligomeric folding intermediates were separated from the monomer by native gel electrophoresis, the reduction of intermolecular disulfide bonds did not occur in the subsequent second-dimension SDS-gel electrophoresis. This result suggests that when tailspike monomer is present in free solution with SDS, the partially unfolded tailspike monomer can facilitate the reduction of disulfide bonds in the tailspike oligomers.Keywords: P22 tailspike protein (TSP); transient disulfide bond; assembly; disulfide shuffling; SDS P22 tailspike protein is a member of the elongated b-helix family of viral adhesins that participates in polysaccharide binding during viral infection that includes pectate lyase C and pertactin (Mitraki et al 2002;Simkovsky and King 2006). Tailspike is a homotrimer and each monomer has 666 amino acid residues.…”