Kinetically stable proteins, those whose stability is derived from their slow unfolding kinetics and not thermodynamics, are examples of evolution's best attempts at suppressing unfolding. Especially in highly proteolytic environments, both partially and fully unfolded proteins face potential inactivation through degradation and/or aggregation, hence, slowing unfolding can greatly extend a protein's functional lifetime. The prokaryotic serine protease α-lytic protease (αLP) has done just that, as its unfolding is both very slow (t1/2 ∼1 year) and so cooperative that partial unfolding is negligible, providing a functional advantage over its thermodynamically stable homologs, such as trypsin. Previous studies have identified regions of the domain interface as critical to αLP unfolding, though a complete description of the unfolding pathway is missing. In order to identify the αLP unfolding pathway and the mechanism for its extreme cooperativity, we performed high temperature molecular dynamics unfolding simulations of both αLP and trypsin. The simulated αLP unfolding pathway produces a robust transition state ensemble consistent with prior biochemical experiments and clearly shows that unfolding proceeds through a preferential disruption of the domain interface. Through a novel method of calculating unfolding cooperativity, we show that αLP unfolds extremely cooperatively while trypsin unfolds gradually. Finally, by examining the behavior of both domain interfaces, we propose a model for the differential unfolding cooperativity of αLP and trypsin involving three key regions that differ between the kinetically stable and thermodynamically stable classes of serine proteases.

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Ultrahigh-resolution (<1.0 Å) structures have revealed unprecedented and unexpected details of molecular geometry, such as the deformation of aromatic rings from planarity. However, the functional utility of such energetically costly strain is unknown. The 0.83 Å structure of α-lytic protease (αLP) indicated that residues surrounding a conserved Phe side-chain dictate a rotamer which results in a ∼6°distortion along the side-chain, estimated to cost 4 kcal∕mol. By contrast, in the closely related protease Streptomyces griseus Protease B (SGPB), the equivalent Phe adopts a different rotamer and is undistorted. Here, we report that the αLP Phe side-chain distortion is both functional and conserved in proteases with large pro regions. Sequence analysis of the αLP serine protease family reveals a bifurcation separating those sequences expected to induce distortion and those that would not, which correlates with the extent of kinetic stability. Structural and folding kinetics analyses of family members suggest that distortion of this side-chain plays a role in increasing kinetic stability within the αLP family members that use a large Pro region. Additionally, structural and kinetic folding studies of mutants demonstrate that strain alters the folding free energy landscape by destabilizing the transition state (TS) relative to the native state (N). Although side-chain distortion comes at a cost of foldability, it suppresses the rate of unfolding, thereby enhancing kinetic stability and increasing protein longevity under harsh extracellular conditions. This ability of a structural distortion to enhance function is unlikely to be unique to αLP family members and may be relevant in other proteins exhibiting side-chain distortions.protein distortion | protein structure | X-ray crystallography | protein stability | protein lifetime T he physicochemical forces that determine macromolecular structure and function are generally thought to be well understood. The predominant forces include well-studied hydrogen bonding, van der Waals, and ionic interactions. By contrast, bonded terms are largely ignored as it is commonly held that bond lengths and angles are at or very close to canonical values in the native state. This view is so widely adopted that nearly all crystallographic and NMR structure determination methods include restraints to optimal values. However, distortion of covalent bonds in protein sidechains away from their low energy configurations has now been noted in several ultrahigh-resolution structures (1, 2) (Fig. 1A), but no functional role for the distortion has been ascribed. We examine the functional consequences of side-chain distortion in α-lytic protease (αLP), in which a 0.83 Å structure (2) revealed a surprisingly large distortion of the aromatic ring of a conserved Phe (Phe228) by almost 6°from planarity ( Fig. 1B; about 2.6 standard deviations from the mean in Fig. 1A).The αLP sub-family of serine proteases is one of the best-studied examples of a kinetically stable protein (3, 4); i.e. a protein that...