The Murine Orthologue of Human Antichymotrypsin
2005; Elsevier BV; Volume: 280; Issue: 52 Linguagem: Inglês
10.1074/jbc.m505598200
ISSN1083-351X
AutoresAnita J. Horvath, James A. Irving, Jamie Rossjohn, Ruby H. P. Law, Stephen Bottomley, Noelene S. Quinsey, Robert N. Pike, Paul Coughlin, James C. Whisstock,
Tópico(s)Alzheimer's disease research and treatments
ResumoAntichymotrypsin (SERPINA3) is a widely expressed member of the serpin superfamily, required for the regulation of leukocyte proteases released during an inflammatory response and with a permissive role in the development of amyloid encephalopathy. Despite its biological significance, there is at present no available structure of this serpin in its native, inhibitory state. We present here the first fully refined structure of a murine antichymotrypsin orthologue to 2.1 Å, which we propose as a template for other antichymotrypsin-like serpins. A most unexpected feature of the structure of murine serpina3n is that it reveals the reactive center loop (RCL) to be partially inserted into the A β-sheet, a structural motif associated with ligand-dependent activation in other serpins. The RCL is, in addition, stabilized by salt bridges, and its plane is oriented at 90° to the RCL of antitrypsin. A biochemical and biophysical analysis of this serpin demonstrates that it is a fast and efficient inhibitor of human leukocyte elastase (ka: 4 ± 0.9 × 106 m-1 s-1) and cathepsin G (ka: 7.9 ± 0.9 × 105 m-1 s-1) giving a spectrum of activity intermediate between that of human antichymotrypsin and human antitrypsin. An evolutionary analysis reveals that residues subject to positive selection and that have contributed to the diversity of sequences in this sub-branch (A3) of the serpin superfamily are essentially restricted to the P4–P6′ region of the RCL, the distal hinge, and the loop between strands 4B and 5B. Antichymotrypsin (SERPINA3) is a widely expressed member of the serpin superfamily, required for the regulation of leukocyte proteases released during an inflammatory response and with a permissive role in the development of amyloid encephalopathy. Despite its biological significance, there is at present no available structure of this serpin in its native, inhibitory state. We present here the first fully refined structure of a murine antichymotrypsin orthologue to 2.1 Å, which we propose as a template for other antichymotrypsin-like serpins. A most unexpected feature of the structure of murine serpina3n is that it reveals the reactive center loop (RCL) to be partially inserted into the A β-sheet, a structural motif associated with ligand-dependent activation in other serpins. The RCL is, in addition, stabilized by salt bridges, and its plane is oriented at 90° to the RCL of antitrypsin. A biochemical and biophysical analysis of this serpin demonstrates that it is a fast and efficient inhibitor of human leukocyte elastase (ka: 4 ± 0.9 × 106 m-1 s-1) and cathepsin G (ka: 7.9 ± 0.9 × 105 m-1 s-1) giving a spectrum of activity intermediate between that of human antichymotrypsin and human antitrypsin. An evolutionary analysis reveals that residues subject to positive selection and that have contributed to the diversity of sequences in this sub-branch (A3) of the serpin superfamily are essentially restricted to the P4–P6′ region of the RCL, the distal hinge, and the loop between strands 4B and 5B. α1-Antichymotrypsin (SERPINA3) 6The abbreviations used are: SERPINA3α1-antichymotrypsin (referred to herein as huACT)Cat Gcathepsin GRCLreactive center loophuAThuman antitrypsinHLEHuman leukocyte elastasepNAp-nitroanilideSIstoichiometry of inhibitionr.m.s.d.root mean square deviation.6The abbreviations used are: SERPINA3α1-antichymotrypsin (referred to herein as huACT)Cat Gcathepsin GRCLreactive center loophuAThuman antitrypsinHLEHuman leukocyte elastasepNAp-nitroanilideSIstoichiometry of inhibitionr.m.s.d.root mean square deviation. is a member of the serpin superfamily of protease inhibitors. Like its close orthologue α1-antitrypsin, it was first characterized as an acute phase plasma protease inhibitor (1Dickson I. Alper C.A. Clin. Chim. Acta. 1974; 54: 381-385Crossref PubMed Scopus (22) Google Scholar) and is synthesized in a range of tissues, including hepatocytes (2Chandra T. Stackhouse R. Kidd V.J. Robson K.J. Woo S.L. Biochemistry. 1983; 22: 5055-5061Crossref PubMed Scopus (161) Google Scholar), bronchial epithelial cells (3Cichy J. Potempa J. Chawla R.K. Travis J. J. Clin. Invest. 1995; 95: 2729-2733Crossref PubMed Scopus (50) Google Scholar), and neuronal cells (4Hwang S.R. Steineckert B. Kohn A. Palkovits M. Hook V.Y. J. Biol. Chem. 1999; 274: 1821-1827Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). In the lung, α1-antichymotrypsin (referred to as huACT hereafter) plays an important role in the regulation of proteases released by leukocytes during an inflammatory response (5Travis J. Bowen J. Baugh R. Biochemistry. 1978; 17: 5651-5656Crossref PubMed Scopus (132) Google Scholar), cathepsin G (Cat G) (5Travis J. Bowen J. Baugh R. Biochemistry. 1978; 17: 5651-5656Crossref PubMed Scopus (132) Google Scholar), and mast cell chymase (6Schechter N.M. Jordan L.M. James A.M. Cooperman B.S. Wang Z.M. Rubin H. J. Biol. Chem. 1993; 268: 23626-23633Abstract Full Text PDF PubMed Google Scholar). In the brain, huACT has been identified in amyloid plaques (7Abraham C.R. Selkoe D.J. Potter H. Cell. 1988; 52: 487-501Abstract Full Text PDF PubMed Scopus (795) Google Scholar) and its overexpression plays a permissive role in the progression of Alzheimer disease (8Kamboh M.I. Sanghera D.K. Ferrell R.E. DeKosky S.T. Nat. Genet. 1995; 10: 486-488Crossref PubMed Scopus (322) Google Scholar) and cerebral amyloid encephalopathy (9Yamada M. Sodeyama N. Itoh Y. Suematsu N. Otomo E. Matsushita M. Mizusawa H. Ann. Neurol. 1998; 44: 129-131Crossref PubMed Scopus (40) Google Scholar). Current evidence suggests that its role in the brain, and consequently the basis of its association with amyloid plaques, is as a component of the inflammatory response (10Abraham C.R. Neurobiol. Aging. 2001; 22: 931-936Crossref PubMed Scopus (76) Google Scholar). α1-antichymotrypsin (referred to herein as huACT) cathepsin G reactive center loop human antitrypsin Human leukocyte elastase p-nitroanilide stoichiometry of inhibition root mean square deviation. α1-antichymotrypsin (referred to herein as huACT) cathepsin G reactive center loop human antitrypsin Human leukocyte elastase p-nitroanilide stoichiometry of inhibition root mean square deviation. Serpins are the largest family of protease inhibitors and extend to all branches of life (11Irving J.A. Pike R.N. Lesk A.M. Whisstock J.C. Genome Res. 2000; 10: 1845-1864Crossref PubMed Scopus (504) Google Scholar, 12Irving J.A. Steenbakkers P.J.M Lesk A.M. Op den Camp H.J.M Pike R.N. Whisstock J.C.W Mol. Biol. Evol. 2002; 19: 1881-1890Crossref PubMed Scopus (105) Google Scholar). Members of this class of protein perform roles in diverse physiological processes such as the blood clotting cascade, apoptosis, and chromatin condensation (13Silverman G.A. Bird P.I. Carrell R.W. Church F.C. Coughlin P.B. Gettins P.G. Irving J.A. Lomas D.A. Luke C.J. Moyer R.W. Pemberton P.A. Remold-O'Donnell E. Salvesen G.S. Travis J. Whisstock J.C. J. Biol. Chem. 2001; 276: 33293-33296Abstract Full Text Full Text PDF PubMed Scopus (1047) Google Scholar). The serpin fold is highly conserved and consists of 3 β-sheets, 8–9 α-helices, and a solvent-exposed stretch of amino acids termed the reactive center loop (RCL) (14Whisstock J. Skinner R. Lesk A.M. Trends Biochem. Sci. 1998; 23: 63-67Abstract Full Text PDF PubMed Scopus (161) Google Scholar). The mechanism of inhibition has been demonstrated biophysically and structurally. Serpins "hijack" the proteolytic mechanism, during which a protease and substrate are transiently linked by an acyl ester bond. The cognate protease binds to, and cleaves within, specificity-determining residues of the RCL; following cleavage of the peptide bond, the RCL is thermodynamically driven to insert into the center of the A β-sheet, the tethered protease translocates ∼70 Å and becomes compressed against the base of the serpin. Distortion of the active site prevents the final hydrolysis event, and the result is an irreversible, covalent serpin-enzyme complex (15Huntington J.A. Read R.J. Carrell R.W. Nature. 2000; 407: 923-926Crossref PubMed Scopus (932) Google Scholar). The ability of serpins to accommodate a nascent RCL confers an important vulnerability: the propensity to form long chain polymers in which the RCL of one molecule becomes embedded in the A β-sheet (or the C β-sheet) of the next (16Dunstone M.A. Dai W. Whisstock J.C. Rossjohn J. Pike R.N. Feil S.C. Le Bonniec B.F. Parker M.W. Bottomley S.P. Protein Sci. 2000; 9: 417-420Crossref PubMed Scopus (81) Google Scholar, 17Chang W.S. Whisstock J.C. Hopkins P.C. Lesk A.M. Carrell R.W. Wardell M.R. Protein Sci. 1997; 6: 89-98Crossref PubMed Scopus (72) Google Scholar). This state can be brought about by point mutation. For example, the Pro229 → Ala mutation renders huACT susceptible to polymerization: the protein is deposited as inclusions in hepatocytes leading to liver damage, and the resulting deficiency in the lungs results in obstructive pulmonary disease (18Faber J.P. Poller W. Olek K. Baumann U. Carlson J. Lindmark B. Eriksson S. J. Hepatol. 1993; 18: 313-321Abstract Full Text PDF PubMed Scopus (78) Google Scholar). Similarly, the Leu55 → Pro mutation destabilizes the serpin, leading to the formation of polymers and the inactive, monomeric "latent" form (19Poller W. Faber J.P. Weidinger S. Tief K. Scholz S. Fischer M. Olek K. Kirchgesser M. Heidtmann H.H. Genomics. 1993; 17: 740-743Crossref PubMed Scopus (90) Google Scholar). In rats and mice the antichymotrypsin gene has undergone extensive duplication and diversification (20Forsyth S. Horvath A. Coughlin P. Genomics. 2003; 81: 336-345Crossref PubMed Scopus (65) Google Scholar), yielding a family of 13 closely related inhibitors within the murine serpina3 cluster with differing tissue distribution and protease specificity (21Horvath A.J. Forsyth S.L. Coughlin P.B. J. Mol. Evol. 2004; 59: 488-497Crossref PubMed Scopus (27) Google Scholar, 22Inglis J.D. Hill R.E. EMBO J. 1991; 10: 255-261Crossref PubMed Scopus (43) Google Scholar). A similar event has also occurred at some other serpin loci, including antitrypsin (23Barbour K.W. Wei F. Brannan C. Flotte T.R. Baumann H. Berger F.G. Genomics. 2002; 80: 515-522Crossref PubMed Google Scholar) and MNEI, PI-6, and PI-9 (24Kaiserman D. Knaggs S. Scarff K.L. Gillard A. Mirza G. Cadman M. McKeone R. Denny P. Cooley J. Benarafa C. Remold-O'Donnell E. Ragoussis J. Bird P.I. Genomics. 2002; 79: 349-362Crossref PubMed Scopus (54) Google Scholar), and has been observed for numerous genes throughout the mouse and rat genomes (25Cheung J. Wilson M.D. Zhang J. Khaja R. MacDonald J.R. Heng H.H. Koop B.F. Scherer S.W. Genome. Biol. 2003; 4: R47Crossref PubMed Google Scholar). Gene expression studies suggest that serpina3n (also known as EB22.4 and referred to here as muACT-n) is the closest murine orthologue of huACT. In particular, it is the only member of the serpina3 cluster that is expressed in the murine brain under resting conditions (21Horvath A.J. Forsyth S.L. Coughlin P.B. J. Mol. Evol. 2004; 59: 488-497Crossref PubMed Scopus (27) Google Scholar), consistent with evidence that huACT plays a role in the inflammatory response in that organ. As with huACT, muACT-n demonstrates a wide tissue distribution: it is found in the liver, brain, testis, lung, thymus, spleen, and to a lesser extent bone marrow, skeletal muscle, and kidney (21Horvath A.J. Forsyth S.L. Coughlin P.B. J. Mol. Evol. 2004; 59: 488-497Crossref PubMed Scopus (27) Google Scholar). Because muACT-n is a likely orthologue of huACT, we sought to characterize this protein both structurally and kinetically. We demonstrate that muACT-n is expressed as a functional, inhibitory serpin and possesses activity that bridges the specificities of both huACT and human antitrypsin (huAT). The structure of this serpin reveals that the RCL is partially inserted into the A β-sheet, is tilted almost 90° with respect to huAT and antithrombin, and is stabilized by two salt bridges tethering it to the body of the serpin. The positively charged residues involved in DNA binding in huACT were also found to be solvent-exposed muACT-n and contribute to one of two patches of positive charge (near the s3C–s4C loop and on the F-helix) on one face of the serpin. These data provide a framework for developing a further understanding of the role of the murine a3 serpins in the biology of their host. Reagents—Restriction enzymes, T4 DNA ligase, and Vent polymerase were purchased from New England Biolabs. Superscript II reverse transcriptase was obtained from Invitrogen. Bovine chymotrypsin and trypsin were purchased from Sigma-Aldrich. Human leukocyte elastase (HLE) and human leukocyte Cat G were obtained from Athens Research Technology (Athens, GA). Unless otherwise stated general chemicals were purchased from Sigma, and chromatographic equipment was purchased from Bio-Rad. Colorimetric assays were performed using a Thermomax microplate reader from Molecular Devices (Sunnyvale, CA). Production of Recombinant muACT-n—Mouse brain was isolated from 7-week NMRI mice, and RNA was extracted using the acid-guanidinium/phenol/chloroform method (26Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar). 2 μg of total RNA was reverse-transcribed with Superscript II reverse transcriptase and 5 pmol/liter oligo(dT). For expression of muACT-n, the sense primer was designed to exclude the predicted N-terminal secretion peptide. Primers introducing BamHI restriction site tails for cloning into the pET-His(3a) expression vector were as follows: sense (5′-ATGGATCCTTCCCAGATGGCAC-3′) and antisense (5′-ATGGATCCTCATTTGGGGTTGGCT-3′). The muACT-n was subcloned into the BamHI restriction site of the pET-His(3a) expression vector to produce an N-terminal His-tagged protein. Correct orientation and sequence were confirmed by DNA sequencing. The construct was then used to transform BL21(DE3) pLysS cells and recombinant protein production induced with 0.2 mm isopropyl 1-thio-β-d-galactopyranoside (final concentration) during the mid-log growth phase for 2 h. Cells were harvested by centrifugation (1,500 × g) and resuspended in lysis buffer (50 mm sodium phosphate, pH 7.8, 100 mm NaCl, 10 mm imidazole) to which 1 mg/ml lysozyme, 20 μg/ml phenylmethylsulfonyl fluoride, protease inhibitor mixture (1/1000), and 5 mm 2-β-mercaptoethanol was added. The lysate was incubated on ice for 30 min and 25 μg/ml DNase was added. The cells were freeze/thawed three times in liquid N2 and a 37 °C water bath, respectively. Soluble protein was bound to footnote as nickel-nitrilotriacetic acid-agarose (Qiagen), eluted with a 0.125 m imidazole step gradient, and further purified using a Mono Q column (Amersham Biosciences) with a 0—0.5 m NaCl gradient in 20 mm Tris, pH 8.0, 0.1 mm EDTA, and 5 mm 2-β-mercaptoethanol. Digestion of muACT-n by Trypsin—To generate RCL-cleaved serpin, 1 mg of muACT-n was incubated with trypsin at 20:1 (w:w) at 37 °C for 2 h. The reactions were stopped by the addition of phenylmethylsulfonyl fluoride to a final concentration of 20 μg/ml and samples placed on ice. Cleaved proteins were purified on a Mono Q column to remove traces of protease. Circular Dichroism—Circular dichroism analysis was performed on a Jasco 820s spectropolarimeter (Jasco, Easton, MD). Changes in protein secondary structure were monitored by measuring the change in ellipticity at 222 nm using a 0.05-cm path-length cuvette and a protein concentration of 0.2 mg/ml in phosphate buffered saline, pH 7.4. Thermal unfolding experiments were performed by heating at a rate of 1 °C/min from 25 to 90 °C. Complex Formation Assays—The ability of muACT-n to inhibit the serine proteases bovine chymotrypsin and trypsin, Cat G, HLE, human plasma thrombin, and factor Xa was investigated by SDS-PAGE. 1 μm of each serine protease was incubated with either 1 μm or 5 μm muACT-n in 1× phosphate-buffered saline. Reactions were incubated at 37 °C for 30 min, then immediately placed on ice, and SDS-PAGE reducing buffer was added. Samples were denatured at 95 °C for 5 min, separated on 10% reducing SDS-PAGE, and transferred to polyvinylidene difluoride by Western blotting. Membranes were probed with mouse-His-antibody (1/2000) and goat-anti-mouse-horseradish peroxidase (1/5000). Proteins were detected with ECL reagent and visualized by autoradiography. Stoichiometry of Inhibition—Stoichiometry of inhibition (SI) values were determined for the interaction between with chymotrypsin, Cat G, or HLE and muACT-n. muACT-n (0.5–4 nm) was incubated with a constant concentration of chymotrypsin (2 nm) at 37°C for 4 h, and residual activity was assayed with the substrate N-succinyl-Ala-Ala-Pro-Phe-pNA (100 μm). Inhibition of Cat G (30 nm) was titrated with increasing concentrations of muACT-n (7.5–60 nm) at room temperature for 2 h, and the residual activity was assayed with the substrate N-succinyl-Ala-Ala-Pro-Phe-pNA (500 μm) on bovine serum albumin-coated microtiter plates. HLE (2 nm) was incubated with increasing concentrations of muACT-n (0.5–4 nm) at 37 °C for 2 h and assayed with 200 μm N-methoxy-Ala-Ala-Pro-Val-pNA on bovine serum albumin-coated microtiter plates. Following linear regression analysis of the plot of residual enzyme activity against serpin concentration, the SI was determined by extrapolating to the protease:serpin ratio where protease activity is zero. The SI values represent the average of three separate experiments. Association Rate Constant (Continuous Method)—Rate constants were measured under pseudo-first order conditions using the progress-curve method (27Le Bonniec B.F. Guinto E.R. Stone S.R. Biochemistry. 1995; 34: 12241-12248Crossref PubMed Scopus (48) Google Scholar) for the interaction of muACT-n with HLE and Cat G. For HLE, assays were carried out using 0.4 nm HLE, 0.4–2.8 nm muACT-n, and 200 μm N-methoxy-Ala-Ala-Pro-Val-pNA at 37 °C in bovine serum albumin coated microtiter plates. For cat G, assays were performed with 20 nm Cat G, 4–40 nm muACT-n, and 1 mm N-succinyl-Ala-Ala-Pro-Phe-pNA at room temperature in bovine serum albumin-coated microtiter plates. A constant amount of protease was mixed with varying amounts of muACT-n and substrate, with the rate of product formation (measured at A405 nm) described by Equation 1,P=V0kobs×[1-e(-kobst)](Eq. 1) where P is the concentration of product at time t, kobs is the apparent first-order rate constant and vo is the initial velocity (28Rovelli G. Stone S.R. Guidolin A. Sommer J. Monard D. Biochemistry. 1992; 31: 3542-3549Crossref PubMed Scopus (40) Google Scholar), for each inhibitor concentration [I], a kobs value was calculated by non-linear least-squares fitting of the data to Equation 1. The resulting kobs values were plotted against [I], and the uncorrected second-order rate constant (k′) determined from the slope of the line of best fit; k′ was corrected for substrate concentration [S], the Km of the protease and SI yielding the second-order rate constant, ka (Equation 2) as follows.ka=k'×(1+([S]/Km)×SI(Eq. 2) The ka values for the inhibition of Cat G and HLE represent the average of two and three separate experiments, respectively. Regression analysis was performed using GraphPad Prism 3 (GraphPad Software, San Diego, CA). Association Rate Constant (Discontinuous Method)—A discontinuous method (29Olson S.T. Bjork I. Shore J.D. Methods Enzymol. 1993; 222: 525-559Crossref PubMed Scopus (260) Google Scholar) was used to determine the rate of inhibition (ka) of chymotrypsin by muACT-n. The pseudo-first order rate constant with chymotrypsin (2 nm) and muACT-n (10–100 nm) was determined by incubation for different periods of time (0–5 min) followed by measurement of residual chymotrypsin activity. The pseudo-first order constant, kobs, was determined from the slope of a semi-log plot of the residual protease activity against time. The kobs values were then plotted against serpin concentration and the slope of the line of best fit gave an estimate of the second-order rate constant, ka. The ka values represent the average of three separate experiments. Crystallization of muACT-n—Crystallization trials were undertaken using the hanging drop vapor diffusion technique, employing a 1-μl: 1-μl drop ratio of muACT-n (at 15 mg/ml) to mother liquor, with a 0.5- to 1-ml reservoir volume at 22 °C. Single large crystals grew in 24% polyethylene glycol 3350, 0.1 m sodium tartrate, 0.1 m HEPES, pH 6.6, after 2 weeks. The crystals were flash-cryocooled prior to data collection with 15% glycerol as the cryoprotectant. X-ray Data Collection, Structure Determination, and Refinement—Out of several crystals screened, one diffracted to 2.1 Å resolution; integration and scaling using the HKL suite (30Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar) revealed it belonged to space group P212121, with unit cell dimensions of a = 83.34 Å, b = 92.62 Å, and c = 118.26 Å. This is consistent with two monomers in the asymmetric unit and ∼52% solvent content as predicted using the CCP4 program Matthews (31CCP4 Acta Crystallogr. D. Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar). TABLE ONE presents a summary of data collection statistics.TABLE ONEComparison of inhibitory kinetics of muACT-n, huACT, and huATProteaseSerpinSIkaRef.m–1 s–1Chymotrypsin (bovine)muACT-n25.8 ± 0.7×104huACT6×104(54Beatty K. Bieth J. Travis J. J. Biol. Chem. 1980; 255: 3931-3934Abstract Full Text PDF PubMed Google Scholar)15.5×105(55Lomas D.A. Stone S.R. Llewellyn-Jones C. Keogan M.T. Wang Z.M. Rubin H. Carrell R.W. Stockley R.A. J. Biol. Chem. 1995; 270: 23437-23443Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar)5.9×106(54Beatty K. Bieth J. Travis J. J. Biol. Chem. 1980; 255: 3931-3934Abstract Full Text PDF PubMed Google Scholar)huAT13.6×106(55Lomas D.A. Stone S.R. Llewellyn-Jones C. Keogan M.T. Wang Z.M. Rubin H. Carrell R.W. Stockley R.A. J. Biol. Chem. 1995; 270: 23437-23443Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar)Cathepsin G (human)muACT-n17.9 ± 0.9×1055.1×107(54Beatty K. Bieth J. Travis J. J. Biol. Chem. 1980; 255: 3931-3934Abstract Full Text PDF PubMed Google Scholar)huACT18.1×105(55Lomas D.A. Stone S.R. Llewellyn-Jones C. Keogan M.T. Wang Z.M. Rubin H. Carrell R.W. Stockley R.A. J. Biol. Chem. 1995; 270: 23437-23443Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar)7.0×105(56Duranton J. Adam C. Bieth J.G. Biochemistry. 1998; 37: 11239-11245Crossref PubMed Scopus (56) Google Scholar)huAT1.14.1×105(54Beatty K. Bieth J. Travis J. J. Biol. Chem. 1980; 255: 3931-3934Abstract Full Text PDF PubMed Google Scholar)Leukocyte elastase (human)muACT-n1.14.0 ± 0.9×106huACTSubstrate(55Lomas D.A. Stone S.R. Llewellyn-Jones C. Keogan M.T. Wang Z.M. Rubin H. Carrell R.W. Stockley R.A. J. Biol. Chem. 1995; 270: 23437-23443Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar)huAT1.15×107(54Beatty K. Bieth J. Travis J. J. Biol. Chem. 1980; 255: 3931-3934Abstract Full Text PDF PubMed Google Scholar)1.9×107(55Lomas D.A. Stone S.R. Llewellyn-Jones C. Keogan M.T. Wang Z.M. Rubin H. Carrell R.W. Stockley R.A. J. Biol. Chem. 1995; 270: 23437-23443Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) Open table in a new tab The structure of muACT-n was solved using the molecular replacement method (resolution range 10 to 3.5 Å) implemented within the MOLREP program (32Vagin A. Teplyakov A. Acta Crystallogr. D. Biol. Crystallogr. 2000; 56: 1622-1624Crossref PubMed Scopus (689) Google Scholar). The structure of human native huAT (pdb accession, 1QLP) proved to be superior as a search model to cleaved huACT (pdb accession, 1AS4), providing tentative evidence that the molecules within the crystal corresponded more closely with the native conformation. In both search models, the sequence differences had been mutated to alanine. A single peak in rotation and two strong peaks in the translation function revealed two molecules separated by an orthogonal pseudo-translation of (49.7, -2.5, and 2.9 Å). The molecules packed well within the unit cell and the resulting initial electron density map revealed strong unbiased features, particularly in the region of the RCL, suggesting the molecular replacement had been successful. The progress of refinement (using data between 2.1 and 30 Å) was monitored by the Rfree value without a sigma cut-off applied to the data. The structure was refined in CNS version 1.1 (33Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. D. Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar) using successive rounds of: 1) rigid-body fitting of the individual domains; 2) imposition of strict non-crystallographic symmetry constraints during simulated annealing and individual B-factor refinement; and 3) manual model building using the program O (34Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard Acta Crystallogr. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). Bulk solvent corrections were applied throughout. Once the Rfree value dropped below ∼35%, the two molecules were treated separately with medium NCS positional (150/Å2) and B-factor (2.5σ) restraints on residues not involved in crystal or NCS contacts. Refinement proceeded with rounds of 1) maximum-likelihood minimization, 2) B-factor refinement, and 3) model building. Water molecules were manually built into the model if they were within hydrogen-bonding distance to chemically reasonable groups, if they appeared in Fo - Fc maps contoured at 3.0) σ, and subsequently generated 2Fo - Fc maps contoured at 1.0 σ. The two final rounds of refinement were undertaken with REFMAC5 (35Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. D. Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar), using default medium main-chain and loose side-chain NCS restraints, individual isotropic B-factors, Babinet solvent scaling, and translation liberation screw refinement. Residue numbering is that of the precursor protein, with the start codon methionine at position 1. Structural Analysis—Hydrogen bonds and salt bridges were calculated using HBPLUS (36McDonald I.K. Thornton J.M. J. Mol. Biol. 1994; 238: 777-793Crossref PubMed Scopus (1850) Google Scholar), surface area using DSSP (37Kabsch W. Sander C. Biopolymers. 1983; 22: 2577-2637Crossref PubMed Scopus (11986) Google Scholar), and superpositions conducted using LSQMAN (38Kleywegt G.J. Jones T.A. Structure. 1995; 3: 535-540Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). MolScript (39Kraulis P.J. J. App. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), Bobscript (40Esnouf R.M. Acta Crystallogr. D Biol. Crystallogr. 1999; 55: 938-940Crossref PubMed Scopus (849) Google Scholar), and Raster3D (41Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3866) Google Scholar) were used to produce structure cartoons, GRASP (42Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5311) Google Scholar) to produce surface potential maps, and Alscript (43Barton G.J. Protein Eng. 1993; 6: 37-40Crossref PubMed Scopus (1108) Google Scholar) to produce the alignment figure. Crystal contacts (cutoff 3.5 Å, replace with SYMBOL) were calculated using the WHATIF server (44Vriend G. J. Mol. Graph. 1990; 8 (29): 52-56Crossref PubMed Scopus (3343) Google Scholar). Sequence Analysis—Antitrypsin and antichymotrypsin-like protein sequences were identified from human, cow, pig, and rat using the BLAST algorithm (45Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. 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Although b failed to resolve the branching arrangement satisfactorily, a and c yielded essentially identical, fairly well resolved trees. The cons
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