Preventing serpin aggregation: the molecular mechanism of citrate action upon antitrypsin unfolding

Abstract

The aggregation of antitrypsin into polymers is one of the causes of neonatal hepatitis, cirrhosis, and emphysema. A similar reaction resulting in disease can occur in other human serpins, and collectively they are known as the serpinopathies. One possible therapeutic strategy involves inhibiting the conformational changes involved in antitrypsin aggregation. The citrate ion has previously been shown to prevent antitrypsin aggregation and maintain the protein in an active conformation; its mechanism of action, however, is unknown. Here we demonstrate that the citrate ion prevents the initial misfolding of the native state to a polymerogenic intermediate in a concentration-dependent manner. Furthermore, we have solved the crystal structure of citrate bound to antitrypsin and show that a single citrate molecule binds in a pocket between the A and B beta-sheets, a region known to be important in maintaining antitrypsin stability.

Figure 1.

Effect of citrate concentration on rate of AAT unfolding. Plots of the logarithm of the initial rate of fluorescence increase (○) and the slower rate of fluorescence decrease (•) versus citrate concentration. The inset shows the unfolding of AAT (1 μM), monitored by rapid dilution into 5.5 M GdnHCl, in the presence (solid line) and absence (dashed line) of 0.5 M citrate. Unfolding was followed by monitoring the changes in intrinsic tryptophan fluorescence (excitation wavelength at 280 nm; emission wavelength at 330 nm).

Figure 2.

Polymerization of AAT in the presence of citrate. (A) AAT was incubated at 60°C in a bis-ANS solution either with (○) or without (•) 0.5 M citrate present. (B) The initial rate of conformational change, k_CC, versus citrate concentration. Each bar represents the average of five separate experiments.

Figure 3.

Diagrammatic representation of the three-dimensional structure of the AAT–citrate complex. The protein is shown in ribbon style, with sheet A in blue, sheet B in red, sheet C in yellow, the reactive center loop in green, and the citrate ion is shown as cyan-colored spheres.

Figure 4.

Analysis of the citrate-binding pocket. Stereoscopic figure of residues contributing to the citrate-binding pocket in (A) crystal form A, (B) crystal form B, and (C) the citrate complex. In all three, the protein is shown as sticks colored by atom type (carbon: green, oxygen: red, nitrogen: blue, sulfur: yellow), with water molecules shown as red spheres. In crystal form B, the chloride ion is shown as an orange sphere (B) while in the citrate complex (B) the citrate is shown as sticks colored by atom type, except that the carbon atoms are in cyan. Comparison of the three sites shows that minimal rearrangement of the site has occurred to adapt to citrate binding.