Unfolding Simulations Reveal the Mechanism of Extreme Unfolding Cooperativity in the Kinetically Stable α-Lytic Protease

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.Author Summary: Proteins, synthesized as linear polymers of amino acids, fold up into compact native states, burying their hydrophobic amino acids into their interiors. Protein folding minimizes the non-specific interactions that unfolded protein chains can make, which include aggregation with other proteins and degradation by proteases. Unfortunately, even in the native state, proteins can partially unfold, opening up regions of their structure and making these adverse events possible. Some proteins, particularly those in harsh environments full of proteases, have evolved to virtually eliminate partial unfolding, significantly reducing their rate of degradation. This elimination of partial unfolding is termed “cooperative,” because unfolding is an all-or-none process. One class of proteins has diverged into two families, one bacterial and highly cooperative and the other animal and non-cooperative. We have used detailed simulations of unfolding for members of each family, α-lytic protease (bacterial) and trypsin (animal) to understand the unfolding pathways of each and the mechanism for the differential unfolding cooperativity. Our results explain prior biochemical experiments, reproduce the large difference in unfolding cooperativity between the families, and point to the interface between α-lytic protease's two domains as essential to establishing unfolding cooperativity. As seen in an unrelated protein family, generation of a cooperative domain interface may be a common evolutionary response for ensuring the highest protein stability.