Inactive Conformation of the Serpin
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Inactive conformation of the serpin a1-antichymotrypsin indicates two-stage insertion of the reactive loop: Implications for inhibitory function and conformational disease Bibek Gooptu†, Bart Hazes†, Wun-Shaing W. Chang‡, Timothy R. Dafforn, Robin W. Carrell, Randy J. Read, and David A. Lomas§ Respiratory Medicine Unit, Department of Medicine and Department of Hematology, University of Cambridge, Wellcome Trust Centre for the Study of Molecular Mechanisms in Disease, Cambridge Institute for Medical Research, Wellcome TrustyMedical Research Council Building, Hills Road, Cambridge, CB2 2XY, United Kingdom Edited by Max F. Perutz, Medical Research Council, Cambridge, United Kingdom, and approved November 2, 1999 (received for review July 29, 1999) The serpins are a family of proteinase inhibitors that play a central mediate has been crystallized and the structure has been solved. It role in the control of proteolytic cascades. Their inhibitory mechanism provides a satisfying explanation for the unusual biochemistry with depends on the intramolecular insertion of the reactive loop into partial insertion of the reactive loop into b-sheet A. Surprisingly b-sheet A after cleavage by the target proteinase. Point mutations however, the lower part of the A-sheet is filled by a b-strand derived within the protein can allow aberrant conformational transitions from residues Asn-163 to Thr-170, which are normally part of the characterized by b-strand exchange between the reactive loop of one F-helix and the connecting loop to strand 3A. This configuration is molecule and b-sheet A of another. These loop-sheet polymers result likely to approximate an intermediate in the generation of aberrant in diseases as varied as cirrhosis, emphysema, angio-oedema, and serpin conformations associated with disease and provides insights thrombosis, and we recently have shown that they underlie an into the inhibitory mechanism of active members of the serpin early-onset dementia. We report here the biochemical characteristics superfamily. BIOCHEMISTRY and crystal structure of a naturally occurring variant (Leu-55–Pro) of the plasma serpin a1-antichymotrypsin trapped as an inactive inter- Materials and Methods mediate. The structure demonstrates a serpin configuration with Expression and Characterization of Leu-55–Pro a1-Antichymotrypsin. partial insertion of the reactive loop into b-sheet A. The lower part of a Leu-55–Pro 1-antichymotrypsin was prepared by PCR mutagen- the sheet is filled by the last turn of F-helix and the loop that links it a esis and confirmed by complete sequencing of the 1-antichymo- to s3A. This conformation matches that of proposed intermediates on a trypsin cDNA. Recombinant wild-type and Leu-55–Pro 1-anti- the pathway to complex and polymer formation in the serpins. In chymotrypsin were expressed in the pzm-S plasmid in Escherichia particular, this intermediate, along with the latent and polymerized coli N4830–1 with a temperature-sensitive promoter as detailed (4, conformations, explains the loss of activity of plasma a -antichymo- 1 5). The cells were lysed in a French press, and a -antichymotrypsin trypsin associated with chronic obstructive pulmonary disease in 1 was purified by Q-Sepharose and DNA chromatography as de- patients with the Leu-55–Pro mutation. scribed by Rubin et al. (4). The eluate then was dialyzed into 50 mM Tris, 5 mM EDTA, pH 7.4, loaded onto a heparin-Sepharose lpha-1-antichymotrypsin is an acute-phase protein and a mem- column (Amersham Pharmacia XK16y40), equilibrated in the Aber of the serine proteinase inhibitor or serpin superfamily. It same buffer, and eluted with a 0–0.4 M gradient of KCl. Purity was is synthesized by the liver and bronchial epithelial cells (1), and confirmed by SDSyPAGE, and protein concentration was deter- plasma levels start to rise within 8 hr of an inflammatory response a mined by Lowry assay. Inhibitory activity, association kinetics, and double within 16 hr (2). The precise role of 1-antichymotryp- nondenaturing and transverse urea gradient PAGE, electron mi- sin is uncertain but it is thought to act as an anti-inflammatory agent croscopy, heat stability assays, and the assessment of the rate of inhibiting chymotrypsin, cathepsin G, mast cell chymase, neutrophil insertion of a synthetic 12-mer reactive loop peptide (Ac-Ser-Glu- chemotaxis, and superoxide production (3–8). Like all members of b a a Ala-Ala-Ala-Ser-Thr-Ala-Val-Val-Ile-Ala) into -sheet A of 1- the serpin superfamily, 1-antichymotrypsin is characterized by a antichymotrypsin were performed as detailed (6). dominant b-sheet A and a mobile reactive loop that acts as a pseudosubstrate for the cognate proteinase (9, 10). After docking a Crystallization and Structure Determination. Leu-55–Pro 1-anti- the loop is cleaved by the proteinase at the P1-P19 bond, and the chymotrypsin eluted from the final column as two distinct peaks acyl-enzyme intermediate is trapped as an irreversible complex by denoted g and d. Crystals of d Leu-55–Pro a -antichymotrypsin insertion of the cleaved loop into the A-sheet (11, 12). The intact 1 were grown by equilibrating a 1-ml solution of protein (10 mgyml in reactive loop of a -antichymotrypsin also can be stably incorpo- 1 50 mM Tris, 50 mM KCl, pH 7.4) with 2 ml of precipitant as a rated into the A-sheet in an inactive latent conformation. This hanging drop over 1 ml of precipitant [20% (wtyvol) PEG 4000, 0.2 conformational transition causes local antiproteinase deficiency, which may be important in the pathogenesis of smoking-induced chronic airflow obstruction (6, 13). Chronic airflow obstruction a This paper was submitted directly (Track II) to the PNAS office. also has been associated with mutations of 1-antichymotrypsin a Data deposition: The atomic coordinates and structure factors have been deposited in the that result in plasma deficiency by the retention of variant 1- Protein Data Bank, www.rcsb.org (PDB ID codes 1qmn and r1qmnsf). antichymotrypsin within hepatocytes (14–16). †B.G. and B.H. contributed equally to this work. We describe here the biochemical characterization of the Leu- ‡ a Present address: President’s Laboratory, National Health Research Institutes, Taipei 115, 55–Pro mutant of 1-antichymotrypsin, which results in plasma Taiwan. deficiency and chronic obstructive pulmonary disease (15). Our §To whom reprint requests should be addressed. data show that this mutant can adopt not only the native and The publication costs of this article were defrayed in part by page charge payment. This article inactive latent conformations identified in other serpins, but also an must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely inactive, stable, polymerogenic intermediate. This unusual inter- to indicate this fact. PNAS u January 4, 2000 u vol. 97 u no. 1 u 67–72 Downloaded by guest on September 29, 2021 trypsin fraction had a similar unfolding profile to wild-type protein whereas the other fraction was resistant to unfolding in up to 8 M urea, as is typical for the latent conformation (Fig. 2C). g a The native species of Leu-55–Pro 1-antichymotrypsin had a 5 6 association kinetics with bovine -chymotrypsin (kass 4.3 3 5 21z 21 5 6 5 0.05 10 M s , Ki 213 4 pM; n 3) that were similar to those of the wild-type protein (2.3 6 0.01 3 105 M21zs21 and 257 6 3 pM), but it was less stable when assessed by thermal stability and g a nondenaturingPAGE(Fig.2D).Native Leu-55–Pro 1-antichymo- a trypsin was also less stable than wild-type 1-antichymotrypsin when incubated at 37°C or 41°C and monitored by inhibitory activity or density of the native band on nondenaturing PAGE (Fig. 2E). This loss of activity was associated with the formation of short chain polymers and aggregates when the protein was visualized by electron microscopy (data not shown). These aggregates dissociated to monomers after heating in 1% (wtyvol) SDS at 100°C for 2 min, which is consistent with polymer formation. The latent component g a Fig. 1. Schematic diagram showing the diffraction data characteristics as a of Leu-55–Pro 1-antichymotrypsin remained resistant to heating function of resolution. The data completeness drops off sharply beyond 2.5 Å as at 100°C and has been shown previously to be inactive as a a result of radiation decay, but the higher-resolution reflections are still useful as proteinase inhibitor (6). indicated by the signal-to-noise ratio. In total 18,805 reflections were measured, d Leu-55–Pro a -antichymotrypsin had a similar electrophoretic yielding 11,373 unique observations between 41.0 and 2.3 Å. The overall multi- 1 mobility on nondenaturing PAGE to recombinant wild-type native plicity-weighted Rmerge (43) is 13.1%, increasing to 48.5% in the highest- a 1-antichymotrypsin (Fig. 2B) and was always inactive as an inhib- resolution shell. a a itor of bovine -chymotrypsin. Inactive variants of 1-antichymo- trypsin typically act as substrates and are cleaved at the exposed M (NH ) SO , 0.1 M NaOAc, pH 4.5] at 18°C for 2–4 weeks. The reactive loop to give a characteristic 4-kDa band shift on SDSy 4 2 4 d a crystals had space group P2 with unit cell dimensions of a 5 41.7 PAGE (25). Incubation of Leu-55–Pro 1-antichymotrypsin with 1 a Å, b 5 122.1 Å, c 5 41.7 Å, b 5 101°. Diffraction data were bovine -chymotrypsin in a 2:1 molar ratio of inhibitor to protein- collected at room temperature from a single crystal on station 7.2 ase resulted in nonspecific protein degradation whereas recombi- at the Daresbury Synchrotron (Warrington, U.K.) by using a nant wild-type protein formed SDS-stable complexes when incu- Mar345 detector and a wavelength of 1.448 Å.