Probing the local conformational change of alpha1-antitrypsin

Abstract

The native form of serpins (serine protease inhibitors) is a metastable conformation, which converts into a more stable form upon complex formation with a target protease. It has been suggested that movement of helix-F (hF) and the following loop connecting to strand 3 of beta-sheet A (thFs3A) is critical for such conformational change. Despite many speculations inferred from analysis of the serpin structure itself, direct experimental evidence for the mobilization of hF/thFs3A during the inhibition process is lacking. To probe the mechanistic role of hF and thFs3A during protease inhibition, a disulfide bond was engineered in alpha(1)-antitrypsin, which would lock the displacement of thFs3A from beta-sheet A. We measured the inhibitory activity of each disulfide-locked mutant and its heat stability against loop-sheet polymerization. Presence of a disulfide between thFs3A and s5A but not between thFs3A and s3A caused loss of the inhibitory activity, suggesting that displacement of hF/thFs3A from strand 5A but not from strand 3A is required during the inhibition process. While showing little influence on the inhibitory activity, the disulfide between thFs3A and s3A retarded loop-sheet polymerization significantly. This successful protein engineering of alpha(1)-antitrypsin is expected to be of value in clinical applications. Based on our current studies, we propose that the reactive-site loop of a serpin glides through between s5A and thFs3A for the full insertion into beta-sheet A while a substantial portion of the interactions between hF and s3A is kept intact.

Figure 1.

Structures of α₁-AT. (A) The structure of native form (1QLP) is represented as a ribbon diagram with the position of six engineered disulfide bonds. Left box, focused side view showing three engineered disulfide bonds between thFs3A and s5A (dotted lines). Right box, focused side view showing two engineered disulfide bonds between thFs3A and s3A (dotted lines) and one engineered disulfide between s3A and s5A (solid line). The balls in the structure indicate mutated residues to cysteine. (B) Structure of α₁-AT–trypsin complex (1EZX) showing that the RSL is inserted between s3A and s5A to form a new β-strand. Structures are color-coded: hF/thFs3A, yellow; s3A, red; s4A, pink; s5A, orange; α₁-AT, blue; trypsin, cyan.

Figure 2.

Nonreducing SDS-PAGE analysis of α₁-AT variants. Four representative double-cysteine mutants were analyzed on nonreducing SDS-polyacrylamide gel. Lanes 1,2, K168C–F189C; lanes 3,4, I169C–Y187C; lanes 5,6, K168C–V337C; lanes 7,8, V185C–V333C; lanes 1,3,5,7, reduced form; lanes 2,4,6,8, oxidized form.

Figure 3.

Activity assay of α₁-AT variants. A fixed amount of PPE was mixed with varying concentrations of each α₁-AT mutant, and the residual protease activity was measured with N-succinyl-(Ala)₃-p-nitroanilide as a substrate. Inhibited fraction of the protease activity vs. molar ratio of α₁-AT to PPE is plotted. (Inset) Plot of inhibitory activity of K168C–V337C with disulfide bond shown with extended x-axis. Slope values were derived from the plot by linear regression analysis, and the inhibitory activity of each mutant was calculated as slope_mutant/slope_wild-type. The resultant activity values are summarized in ▶. ⋄, wild type; •, reduced form of K168C–F189C; ○, oxidized form of K168C–F189C; ▾, reduced I169C–Y187C; ▽, oxidized I169C–Y187C; ▪, reduced K168C–V337C; □, oxidized K168C–V337C; ▵, oxidized K168C–V337C that was re-reduced with DTT.

Figure 4.

Heat stability of wild-type and engineered proteins monitored by non-denaturing PAGE. K168C–F189C and I169C–Y187C proteins in their reduced and oxidized forms as well as the wild-type protein were incubated at 60°C. At each designated time point, an aliquot was removed and analyzed by non-denaturing PAGE. Wild-type and reduced proteins show two bands on non-denaturing PAGE, while oxidized proteins that have no free sulfhydryl group showed one band. This can be explained by the fact that the pK _a value of the sulfhydryl group on the cysteine residue (∼pH 8.3) is close to the pH value of the separating condition (pH 8.8).

Figure 5.

Heat stability of wild-type and engineered proteins monitored by inhibitory activity. The same aliquot as in ▶ was used for activity measurement. Inhibitory activity at each time point relative to the activity at zero time of each protein is plotted as a function of incubation time: ⋄, wild type; •, reduced form of K168C–F189C; ○, oxidized form of K168C–F189C; ▾, reduced I169C–Y187C; ▽, oxidized I169C–Y187C.

Figure 6.

Bis-ANS binding of wild-type and engineered proteins. (A) The fluorescence change of bis-ANS followed by loop–sheet polymerization process of α₁-AT was monitored. (B) The excitation (bandwidth 5 nm) and emission (bandwidth 5 nm) wavelengths used were 420 and 485 nm, respectively. All proteins were incubated in 50 mM Tris-HCl, 50 mM NaCl (pH 8.0), containing bis-ANS.