Probing the folding pathway of a consensus serpin using single tryptophan mutants

Abstract

Conserpin is an engineered protein that represents the consensus of a sequence alignment of eukaryotic serpins: protease inhibitors typified by a metastable native state and a structurally well-conserved scaffold. Previously, this protein has been found to adopt a native inhibitory conformation, possess an atypical reversible folding pathway and exhibit pronounced resistance to inactivation. Here we have designed a version of conserpin, cAT, with the inhibitory specificity of α1-antitrypsin, and generated single-tryptophan variants to probe its folding pathway in more detail. cAT exhibited similar thermal stability to the parental protein, an inactivation associated with oligomerisation rather a transition to the latent conformation, and a native state with pronounced kinetic stability. The tryptophan variants reveal the unfolding intermediate ensemble to consist of an intact helix H, a distorted helix F and ‘breach’ region structurally similar to that of a mesophilic serpin intermediate. A combination of intrinsic fluorescence, circular dichroism, and analytical gel filtration provide insight into a highly cooperative folding pathway with concerted changes in secondary and tertiary structure, which minimises the accumulation of two directly-observed aggregation-prone intermediate species. This functional conserpin variant represents a basis for further studies of the relationship between structure and stability in the serpin superfamily.

The structural basis for Z α 1 -antitrypsin polymerization in the liver

“…It remains to be determined how common or rare the exceptions are to this mechanism among other members of the serpin family. Serpins share a highly conserved core structure and exhibit common folding behaviors, and mutations that are associated with instability and deficiency tend to cluster within defined structural regions ( 37 , 38 ). These factors likely place constraints on the mechanism by which mutations can induce polymerization.…”

Engineering the serpin α1‐antitrypsin: A diversity of goals and techniques

“…Although conserpin was able to inhibit proteinases rapidly, the covalent serpin‐proteinase complexes were not stable . Replacing conserpin RCL residues P7–P1′ with P7‐P2′ from α 1 ‐ AT, so that the RCL length and identity were closer to a natural serpin , improved SI values although complexes continued to dissociate faster than wild type α 1 ‐ AT .…”

“…Although conserpin was able to inhibit proteinases rapidly, the covalent serpin-proteinase complexes were not stable. 89,90 Replacing conserpin RCL residues P7-P1 0 with P7-P2 0 from α 1 -AT, so that the RCL length and identity were closer to a natural serpin, improved SI values although complexes continued to dissociate faster than wild type α 1 -AT. 90,91 Interestingly, the protein remained unable to efficiently inhibit the primary cognate proteinase neutrophil elastase, which may be due to changes in RCL structural dynamics and the modified electrostatic potential of the serpin body.…”

Reactive Centre Loop Dynamics and Serpin Specificity

“…With the aim of changing the specificity of conserpin to that of α1-AT, a conserpin/α1-AT chimera was previously produced 30 , where 9 residues within the RCL (P7-P2') were swapped with the corresponding residues from α1-AT (Fig. 1A).…”

“…Its RCL sequence is sufficiently different from all other serpins such that it no longer resembles an RCL of any serpin with a known target protease. A recent study that investigated the folding pathway of conserpin engineered the P7-P2' sequence of α1-AT into its RCL 30 . The resulting conserpin/α1-AT chimera inhibits chymotrypsin with an SI of 1.46, however, no SI was calculated against HNE.…”