Engineering a Hyperstable Yersinia pestis Outer Membrane Protein Ail Using Thermodynamic Design.

Development of viable therapeutics to effectively combat tier I pneumopathogens such as Yersinia pestis requires a thorough understanding of proteins vital for pathogenicity. The host invasion protein Ail, although indispensable for Yersinia pathogenesis, has evaded detailed characterization, as it is an outer membrane protein with intrinsically low stability and high aggregation propensity. Here, we identify molecular elements of the metastable Ail structure that considerably alter protein-lipid and intraprotein thermodynamics. In addition, we find that four residues Q50, L88, L92, and A94 contribute additively to the lowered stability of Ail, and their conserved substitution is sufficient to re-engineer Ail to Out14, a thermodynamically hyperstable low-aggregation variant with a functional scaffold. Interestingly, Ail also shows two (parallel) folding pathways, which has not yet been reported for ß-barrel membrane proteins. Additionally, we identify the molecular mechanism of enhanced thermodynamic stability of Out14. We show that this enhanced stability of Out14 is due to a favorable change in the nonpolar accessible surface, and the accumulation of a kinetically accelerated off-pathway folding intermediate, which is absent in wild-type Ail. Such engineered hyperstable Ail ß-barrels can be harnessed for targeted drug screening and developing medical countermeasures against Yersiniae. Application of similar strategies will help design effective translational therapeutics to combat biopathogens.

Molecular Switch between Structural Compaction and Thermodynamic Stability by the Xxx-Pro Interface in Transmembrane ß-Barrels.

Transmembrane ß-barrel scaffolds found in outer membrane proteins are formed and stabilized by a defined pattern of interstrand intraprotein H-bonds, in hydrophobic lipid bilayers. Introducing the conformationally constrained proline in ß-barrels can cause significant destabilization of these structural regions that require H-bonding, with proline additionally acting as a secondary structure breaker. Membrane protein ß-barrels are therefore expected to show poor tolerance to the presence of a transmembrane proline. Here, we assign a thermodynamic measure for the extent to which a single proline can be tolerated at the C-terminal interface of the model transmembrane ß-barrel PagP. We find that proline drastically destabilizes PagP by 7.0 kcal mol-1 with respect to wild-type PagP (F161 → P161). Interestingly, strategic modulation of the preceding residue can modify the measured energetics. Placing a hydrophobic or bulky side chain as the preceding residue increases the thermodynamic stability by ≤8.0 kcal mol-1. While polar substituents at the preceding residue decrease the PagP stability, these residues demonstrate stronger tertiary packing interactions in the barrel and retain the catalytic activity of native PagP. This biophysical interplay between enhanced thermodynamic stability and attaining a structurally compact functional ß-barrel scaffold highlights the detrimental effect caused by proline incorporation. Our findings also reveal alternative mechanisms that protein sequences can employ to salvage the structural integrity of transmembrane protein structures.