Engineering an Anion-Binding Cavity in Antichymotrypsin Modulates the “Spring-Loaded” Serpin−Protease Interaction

Abstract

Expressed in a kinetically trapped folding state, a serpin couples the thermodynamic driving force of a massive beta-sheet rearrangement to the inhibition of a target protease. Hence, the serpin-protease interaction is the premier example of a "spring-loaded" protein-protein interaction. Amino acid substitutions in the hinge region of a serpin reactive loop can weaken the molecular spring, which converts the serpin from an inhibitor into a substrate. To probe the molecular basis of this conversion, we report the crystal structure of A349R antichymotrypsin in the reactive loop cleaved state at 2.1 A resolution. This amino acid substitution does not block the beta-sheet rearrangement despite the burial of R349 in the hydrophobic core of the cleaved serpin along with a salt-linked acetate ion. The inhibitory activity of this serpin variant is not obliterated; remarkably, its inhibitory properties are anion-dependent due to the creation of an anion-binding cavity in the cleaved serpin.

Iterative-build OMIT maps: map improvement by iterative model building and refinement without model bias

“…We tested the use of an iterative-build composite OMIT map by applying it to repeat the structure solution of antitrypsin (Kim et al, 2001; PDB code 1hp7) by molecular replacement using the structure of antichymotrypsin (PDB code 1as4; Lukacs et al, 1998) as a search model. We used the PHENIX AutoMR Wizard which calls Phaser (McCoy et al, 2005) to obtain an initial molecular-replacement solution.…”

Design of an allosterically modulated doxycycline and doxorubicin drug-binding protein

“…To achieve this, an arginine was introduced at RCL position P12. Interestingly, the same P12 mutation has been structurally characterized previously in ACT in an entirely different context (22). The authors proposed that β-sheet B serves as a "shock absorber" as a result of the additional space required to accommodate the arginine side chain.…”

“…This is in accordance with our observations in cleaved DBS-I-allo and DBS-II-allo variants with the additional bonus that the doxycycline-and doxorubicin-binding pockets narrow and ligand binding is impeded. A possible route for providing additional energy for disrupting the binding site could be the inclusion of an additional mutation, such as A351R (P10), since the P10 residue also points toward β-sheet B on completion of the S-to-R transition (22). Approximately 10-fold reductions in ligand-binding affinities were observed in DBS-I-allo 1 and DBS-II-allo on RCL cleavage (Table 1).…”

Characterisation of a type II functionally-deficient variant of alpha-1-antitrypsin discovered in the general population

“…A survey of the literature identified a protein engineering study in which the P10 alanine residue of α 1 -antichymotrypsin was arbitrarily mutated to arginine to evaluate its ability to become incorporated into β-sheet A upon cleavage. It was found that due to compensatory molecular rearrangements of the α 1 -antichymotrypsin molecule, there was an increase in substrate behaviour rather than an elimination of inhibitory activity [36]. Correspondingly, we undertook experiments to assess the functional capability of G349R AAT, with a view to ascertaining the clinical impact of this variant.…”

“…An Ala→Arg mutation has been introduced at the equivalent site in a protein engineering study of a related serpin, α 1 -antichymotrypsin [36]. A comparison between the crystal structures of the RCL-cleaved forms of Ala349Arg alpha-1-antichymotrypsin (PDB ID: 1AS4) and the wild-type protein (PDB ID: 2ACH) showed that the introduced arginine side-chain can be accommodated into β-sheet A, but only by rearrangement of the local hydrophobic packing interactions and incorporation of a counter-ion [36]. This is sufficient to greatly reduce inhibitory efficiency, as measured by the non-productive turnover of inhibitor [36].…”