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Pro C domain are important in structuring the protease. This observation is consistent with the fact that smaller related pro regions show sequence homology only to the Pro C domain, thereby maintaining the core structure necessary for binding the ß-hairpin of the protease (Fig. 2b). The positioning of the hairpin and subsequent structuring of the aLP C domain may be a general mechanism for pro region-mediated folding of ß-structures. In contrast, subtilisin, a pro-protease evolutionarily unrelated to aLP, seems to use a different method of pro-catalyzed folding. The subtilisin pro domain stabilizes a pair of a-helices in the protease instead of a ß-hairpin (15). Although aLP and subtilisin have convergently evolved pro-dependent folding, they differ in both their mature protease structures and the method by which their respective pro regions achieve their active protease conformations.

Physical Origins of the Folding Barrier. Although the physical origins of the aLP folding barrier and the means by which Pro lowers this barrier remain to be determined, examination of the enthalpic and entropic contributions to the free-energy difference between the aLP I and N states provides some insights into the nature of the folding barrier. Despite the thermodynamic instability of the dLP N state, titration calorimetry experiments reveal that it is enthalpically favored over the I state by 18 kcal/mol (6). Thus, the thermodynamic stability of the I state must be entropic in origin. This means that either the I and U states are more entropically favored than in “normal” proteins, the aLP N state has lower entropy than normal, or both. One possible source of this excess entropy for I may be the high percentage of glycines found in the aLP sequence. Because glycine residues lack a side chain, they can avoid steric clashes encountered by other amino acids, thereby increasing the number of accessible conformations in the unfolded states. aLP contains 16% glycines compared with only 9% in the homologous but thermodynamically stable chymotrypsin. The 10 additional glycines found in aLP, as compared with chymotrypsin, are predicted to contribute an extra ˜7 kcal/mol of configurational entropy (16) to the unfolded state at 4°C. Removing this additional entropy would be sufficient to alter the direction of the I and N equilibrium, placing the N state at the global free-energy minimum.

The excess unfolding entropy may also be due in part to the extremely low conformational entropy of the aLP native structure. Although native states are often dynamic, aLP adopts a remarkably rigid native structure characterized by insensitivity to proteolysis, unusually low crystallographic B factors, and hydrogen-exchange protection factors on the order of >1010 for ˜40 core amides (J.Davis, J.L.Sohl, and D.A.A., unpublished data). Protection factors of this magnitude have never been observed in any other protein. Contributing to this rigidity, loops in aLP are generally shorter and therefore likely to be less flexible than those found in chymotrypsin. Many of aLP’s extra glycine residues facilitate the tight turns found in these condensed loops. With their ability to assume unusual backbone geometries, the glycines may enable tighter and more cooperative packing within the protein core. In this manner, the high glycine content can reduce the entropy of the N state while increasing the configurational entropy of the I state.

Glycine content appears to be a common feature distinguishing homologous proteases to aLP that have pro regions from those that do not (Table 1). The Streptomyces griseus proteases, along with several other pro region-containing homologues, have 16–20% glycines, whereas the mammalian digestive enzymes and other members of the trypsin serine protease family without pro regions have 6–12% glycines.

Evolution of Longevity Through Kinetic Stability. The correlation between high glycine content and the presence of a conserved ß-hairpin in the protease and the coevolution of a pro region suggests that the rigid native state and large kinetic barrier found in aLP may be conserved in other extracellular bacterial proteases. These shared properties may reflect their common function as proteases that break down microorganisms in the extracellular environment, supplying nutrients for their bacterial hosts. The utility of these proteases is compromised by their tendency to degrade themselves as well as other proteins. As such, it is presumably desirable to the host to evolve proteases that can survive as long as possible under these harsh, degradatory conditions.

A typical protein stabilized thermodynamically without a large barrier preventing unfolding would constantly sample partially and fully unfolded states, leading to rapid destruction by exogenous proteases (Fig. 5a). By contrast, kinetic stability provides a mechanism to increase the cooperativity and raise the barrier to unfolding (Fig. 5b), thereby suppressing breathing motions and global unfolding. The result is a drastic reduction in susceptibility to proteolytic degradation.

Preliminary experiments indicate that this has indeed been a successful strategy for extending aLP’s lifetime when compared with its thermodynamically stabilized homologues chymotrypsin and trypsin. In a survival assay where these three proteases are mixed and allowed to attack each other, aLP retains its biological activity for much longer than its mammalian counterparts (Fig. 5c). The sensitivity of trypsin and chymotrypsin to proteolysis is likely to be a necessary aspect of their regulation in vivo. Additional experiments demonstrate that the rate of aLP autolysis is comparable to the rate of its global unfolding, indicating that transient unfolding motions leading to proteolytic degradation have been suppressed. aLP has been so successfully optimized that it is vulnerable to degradation only after it completely unfolds, which occurs on an extremely slow time scale.

There is a price for kinetic stability, however. The evolution of a large barrier to unfolding and a highly rigid native state through the incorporation of glycines and other changes has, as a consequence, created an even larger barrier to folding and thermodynamically destabilized the native state of aLP. Nature’s solution has been the coevolution of a transient pro region to promote folding by both reducing the folding barrier and stabilizing the native state. Although it is expected that the general principle of longevity through kinetic stability will be shared by the majority of extracellular bacterial proteases and numerous eukaryotic proteases, the precise details of barrier height and degree of thermodynamic destabilization of the native state are likely to vary. aLP, with its large pro region and metastable native state, may be an extreme example.

We thank Dr. Nicholas Sauter for helpful discussions. S.S.J. was supported by a Howard Hughes Medical Institute Predoctoral Fellowship. D.A.A. is an Investigator of the Howard Hughes Medical Institute.

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