Inactive conformation of the serpin alpha(1)-antichymotrypsin indicates two-stage insertion of the reactive loop: implications for inhibitory function and conformational disease

Abstract

The serpins are a family of proteinase inhibitors that play a central role in the control of proteolytic cascades. Their inhibitory mechanism depends on the intramolecular insertion of the reactive loop into beta-sheet A after cleavage by the target proteinase. Point mutations within the protein can allow aberrant conformational transitions characterized by beta-strand exchange between the reactive loop of one molecule and beta-sheet A of another. These loop-sheet polymers result in diseases as varied as cirrhosis, emphysema, angio-oedema, and thrombosis, and we recently have shown that they underlie an early-onset dementia. We report here the biochemical characteristics and crystal structure of a naturally occurring variant (Leu-55-Pro) of the plasma serpin alpha(1)-antichymotrypsin trapped as an inactive intermediate. The structure demonstrates a serpin configuration with partial insertion of the reactive loop into beta-sheet A. The lower part of the sheet is filled by the last turn of F-helix and the loop that links it to s3A. This conformation matches that of proposed intermediates on the pathway to complex and polymer formation in the serpins. In particular, this intermediate, along with the latent and polymerized conformations, explains the loss of activity of plasma alpha(1)-antichymotrypsin associated with chronic obstructive pulmonary disease in patients with the Leu-55-Pro mutation.

Figure 1

Schematic diagram showing the diffraction data characteristics as a function of resolution. The data completeness drops off sharply beyond 2.5 Å as a result of radiation decay, but the higher-resolution reflections are still useful as indicated by the signal-to-noise ratio. In total 18,805 reflections were measured, yielding 11,373 unique observations between 41.0 and 2.3 Å. The overall multiplicity-weighted R_merge (43) is 13.1%, increasing to 48.5% in the highest-resolution shell.

Figure 2

(A) Elution profile of Leu-55–Pro α₁-antichymotrypsin from heparin-Sepharose showing a peak (γ) that elutes at the same KCl concentration as wild-type protein (dashed line) and a higher affinity peak (δ). The peak that elutes near the end of the gradient contains dimeric species. (B) 7.5–15% (wt/vol) nondenaturing PAGE of recombinant wild-type α₁-antichymotrypsin (lane 1), recombinant γ Leu-55–Pro α₁-antichymotrypsin (lane 2), recombinant δ Leu-55–Pro α₁-antichymotrypsin (lane 3). Lanes 1 and 3 contain 7.5 μg protein and lane 2 contains 20 μg to give similar band intensities. γ Leu-55–Pro α₁-antichymotrypsin migrated as two bands with the electrophoretic mobility of native (N) and latent (L) α₁-antichymotrypsin. δ Leu-55–Pro α₁-antichymotrypsin had a similar mobility to recombinant wild-type α₁-antichymotrypsin with a high molecular mass dimer (D). (C) γ Leu-55–Pro α₁-antichymotrypsin (Upper) showed native and latent unfolding transitions on transverse urea gradient PAGE whereas the δ protein (Lower) unfolded with an intermediate profile at approximately 4 M urea. The left of each gel represents 0 M urea and the right 8 M urea. Each gel was loaded with 20 μg protein, which was visualized by silver staining. (d) 7.5–15% (wt/vol) nondenaturing PAGE assessing the thermal stability of wild-type (Top), γ Leu-55–Pro α₁-antichymotrypsin (Middle), and δ Leu-55–Pro α₁-antichymotrypsin (Bottom). Samples were incubated at 0.3 mg/ml for 30 min at the temperatures indicated, and 7.5 μg protein was loaded in each lane. (E) The native conformation of γ (Upper) and δ (Lower) Leu-55–Pro α₁-antichymotrypsin were less stable (as evidenced by the loss of bands on nondenaturing PAGE) than the wild-type protein when incubated under physiological conditions. The samples were incubated at 0.25 mg/ml in 50 mM Tris, 50 mM KCl, pH 7.4 at 41°C for the times indicated and then assessed by 7.5–15% (wt/vol) nondenaturing PAGE. All lanes contain 7.5 μg protein.

Figure 3

(a) Structure of δ Leu-55–Pro α₁-antichymotrypsin showing the reactive loop in red, the A-sheet in green, and the F-helix in yellow. Asn-163, whose side chain mimics the peptide plane in position P₁₁ of s4A, is shown in ball-and-stick representation. Residues 353–357 (P₆-P₂) could not be built and are illustrated as a broken black line. The gate region including s3C and s4C, over which the loop must pass to form the latent conformation, is indicated. The shutter domain underlying the opening of the A-sheet is shown, together with (in black) the five residues at its focus at the s6B-hB junction (Inset). The Leu-55–Pro mutation is shown in red with, in blue, six other mutations that result in serpin polymerization and diseases as diverse as cirrhosis, thrombosis, angio-oedema, and dementia. (b) Cleaved reactive loop of α₁-antichymotrypsin in black (20) with final model of δ Leu-55–Pro α₁-antichymotrypsin shown in yellow. The sigmaa-weighted 2F_o-F_c (purple) and F_o-F_c (pink) electron density maps are shown before rebuilding the molecular replacement model. The break in s4A (between P₁₂ and P₁₀), the connection to helix F at P₁₀, and the density as the loop leaves the A-sheet at position 12 are clearly visible. Clear F_o-F_c density (pink) at position P₇ indicates the presence of Arg-166. (c) β-sheet C from δ Leu-55–Pro α₁-antichymotrypsin (white) superimposed on reactive loop cleaved α₁-antichymotrypsin (black) showing displacement of the main-chain residues of s1C. (d) Schematic illustration of conformational transitions of the serpins. The active serpin (M) has an exposed reactive center loop (red) similar to that of α₁-antitrypsin (30). After activation by heat or destabilizing mutations in the shutter domain (red circle) there is opening of β-sheet A and reactive loop insertion to P₁₂. This state, computationally modeled as a chimera between δ and cleaved α₁-antichymotrypsin, is illustrated as M*. The A-sheet can receive the loop of another molecule (black dotted arrow) to form a loop-sheet dimer (P) and then extend to form a chain of polymers. Alternatively if there is sufficient energy to displace s1C the serpin loop can fully insert to form the latent conformation (L) (29). δ Leu-55–Pro α₁-antichymotrypsin represents a third conformation in which the open A-sheet is filled by insertion of an unfolded loop of the F-helix (yellow). The figures were prepared with molscript (44).

Figure 4

Structure-based sequence alignment of P₉-P₁ of the reactive loop (s4A) of α₁-antichymotrypsin (above) and the strand of the F-helix (hF) that inserts into the A-sheet in our structure (below). Complete (**) or partial (*) conservation of residues in the reactive loop and hF of α₁-antichymotrypsin are shown (conserved) as is the conservation of residues of hF throughout the serpin superfamily (consensus) (41).