C1 Inhibitor Serpin Domain Structure Reveals the Likely Mechanism of Heparin Potentiation and Conformational Disease

C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded β-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-proteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel "sandwich" mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack. C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded β-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-proteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel "sandwich" mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack. C1 inhibitor belongs to the group of serpin-type proteinase inhibitors in blood plasma. Serpins act as pseudosubstrates of serine and cysteine proteinases (although there are noninhibitory serpins as well) (1Gettins P.G.W. Chem. Rev. 2002; 102: 4751-4803Crossref PubMed Scopus (997) Google Scholar). A conformational change is triggered in the serpin upon peptide bond cleavage, which distorts the active site of proteinase and traps it in an inactive, covalently linked serpin-enzyme complex (2Huntington J.A. Read R.J. Carrell R.W. Nature. 2000; 407: 923-926Crossref PubMed Scopus (948) Google Scholar, 3Dementiev A. Dobó J. Gettins P.G.W. J. Biol. Chem. 2006; 281: 3452-3457Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Serpins are vital down-regulator components of proteolytic signal amplification cascades. Human C1 inhibitor (C1-inh) 4The abbreviations used are: C1-inh, C1 inhibitor; rC1-inh, recombinant C1 inhibitor; HAE, hereditary angioedema; GAG, glycosaminoglycan; RCL, reactive center loop; MOPS, 3-(N-morpholino)propanesulfonic acid; fXIIa and fXIa, factor XIIa and XIa, respectively. is the only inhibitor that acts on early components of the classical pathway (C1r and C1s) and on that of the lectin pathway (MASP-1 and MASP-2) of the complement system (4Bos I.G.A. Hack C.E. Abrahams J.P. Immunobiology. 2002; 205: 518-533Crossref PubMed Scopus (52) Google Scholar). Complement mediates host defense against pathogens and altered host cells, but its uncontrolled activation could be harmful. Other physiologically crucial targets include plasma kallikrein and activated factor XII (fXIIa) of the contact activation and activated factor XI (fXIa) of the intrinsic coagulation systems (4Bos I.G.A. Hack C.E. Abrahams J.P. Immunobiology. 2002; 205: 518-533Crossref PubMed Scopus (52) Google Scholar). Apart from proteinase inhibition, it was recently discovered that C1-inh binds the central component C3b of complement (5Jiang H. Wagner E. Zhang H. Frank M.M. J. Exp. Med. 2001; 194: 1609-1616Crossref PubMed Scopus (131) Google Scholar), endotoxins from bacteria (6Liu D. Cai S. Gu X. Scafidi J. Wu X. Davis A.E. II I J. Immunol. 2003; 171: 2594-2601Crossref PubMed Scopus (69) Google Scholar) and E-, P-selectin adhesion proteins on endothelial cells (7Cai S. Davis A.E. J. Immunol. 2003; 171 (3rd.): 4786-4791Crossref PubMed Scopus (81) Google Scholar). C1-inh is a single-chain glycoprotein that has atypical two-domain architecture (8Odermatt E. Berger H. Sano Y. FEBS Lett. 1981; 131: 283-285Crossref PubMed Scopus (27) Google Scholar) with the C-terminal serpin and the unique N-terminal domains (9Bock S.C. Skriver K. Nielsen E. Thøgersen H-C. Wiman B. Donaldson V.H. Eddy R.L. Marrinan J. Radziejewska E. Huber R. Shows T.B. Magnusson S. Biochemistry. 1986; 25: 4292-4301Crossref PubMed Scopus (268) Google Scholar). C1-inh is extensively modified post-translationally, bearing six N-linked carbohydrates. Sequencing analysis revealed 14 potential O-glycosylation sites (9Bock S.C. Skriver K. Nielsen E. Thøgersen H-C. Wiman B. Donaldson V.H. Eddy R.L. Marrinan J. Radziejewska E. Huber R. Shows T.B. Magnusson S. Biochemistry. 1986; 25: 4292-4301Crossref PubMed Scopus (268) Google Scholar), seven of which had been verified by carbohydrate analysis (10Perkins S.J. Smith K.F. Amatayakul S. Ashford D. Rademacher T.W. Dwek R.A. Lachmann P.J. Harrison R.A. J. Mol. Biol. 1990; 214: 751-763Crossref PubMed Scopus (48) Google Scholar). Most of the sugars are present in the N-terminal domain and do not affect proteinase inhibition (11Coutinho M. Aulak K.S. Davis A.E. II I J. Immunol. 1994; 153: 3648-3654PubMed Google Scholar, 12Bos I.G.A. Lubbers Y.T.P. Roem D. Abrahams J.P. Hack C.E. Eldering E. J. Biol. Chem. 2003; 278: 29463-29470Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), but affinity to endotoxins and selectins depends on the N-glycans. The importance of C1-inh is underlined by its deficiency, resulting in hereditary angioedema (HAE) (13Bowen T. Cicardi M. Farkas H. Bork K. Kreuz W. Zingale L. Varga L. Martinez-Saguer I. Aygoören-Puörsuön E. Binkley K. Zuraw B. Davis A. II I Hebert J. Ritchie B. Burnham J. Castaldo A. Menendez A. Nagy I. Harmat G. Bucher C. Lacuesta G. Issekutz A. Warrington R. Yang W. Dean J. Kanani A. Stark D. McCusker C. Wagner E. Rivard G-E. Leith E. Tsai E. MacSween M. Lyanga J. Serushago B. Leznoff A. Waserman S. de Serres J. J. Allergy Clin. Immunol. 2004; 114: 629-637Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Symptoms of HAE manifest themselves in recurrent tissue swelling, which could be lethal if it occurs in the upper airways. Replacement therapy using C1-inh isolated from human plasma was introduced over 25 years ago (14Gadek J.E. Hosea S.W. Gelfand J.A. Santaella M. Wickerhauser M. Triantaphyllopoulos D.C. Frank M.M. N. Engl. J. Med. 1980; 302: 542-546Crossref PubMed Scopus (194) Google Scholar). Its potential as an anti-inflammatory drug was also apparent. Application of C1-inh was found to be beneficial in ischemia-reperfusion injury (organ transplant rejection, heart attack) and septic shock (15Caliezi C. Wuillemin W.A. Zeerleder S. Redondo M. Eisele B. Hack C.E. Pharmacol. Rev. 2000; 52: 91-112PubMed Google Scholar). Recent advances of biotechnology led to the large scale production of recombinant C1-inh from the milk of transgenic rabbits (16van Doorn M.B.A. Burggraaf J. van Dam T. Eerenberg A. Levi M. Hack C.E. Schoemaker R.C. Cohen A.F. Nuijens J. J. Allergy Clin. Immunol. 2005; 116: 876-883Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Activities of many serpins are modulated by ligand binding. The anti-complement activity of heparin, mediated by C1-inh, was recognized decades ago (17Caughman G.B. Boackle R.J. Vesely J. Mol. Immunol. 1982; 19: 287-295Crossref PubMed Scopus (67) Google Scholar), but the mechanism is still unclear (4Bos I.G.A. Hack C.E. Abrahams J.P. Immunobiology. 2002; 205: 518-533Crossref PubMed Scopus (52) Google Scholar, 18Bos I.G.A.C. C1-Inhibitor potentiation by glycosaminoglycans. 2003; (, CIP-Gegevens Koninklijke Bibliotheek, The Hague, Netherlands)Google Scholar). Heparin is a naturally occurring sulfated polysaccharide (glycosaminoglycan (GAG)). The widely used anticoagulant therapy is based on the prototype of GAG-protein interactions, where a heparin chain binds both the serpin antithrombin and the proteinase thrombin. Heparin works as a template; it speeds up the formation of the Michaelis complex and stabilizes it by bridging the two proteins (19Li W. Johnson D.J.D. Esmon C.T. Huntington J.A. Nat. Struct. Mol. Biol. 2004; 11: 857-862Crossref PubMed Scopus (310) Google Scholar, 20Dementiev A. Petitou M. Herbert J-M. Gettins P.G.W. Nat. Struct. Mol. Biol. 2004; 11: 863-867Crossref PubMed Scopus (126) Google Scholar). Allostery also plays an important role in the heparin activation of anti-thrombin (21Johnson D.J.D. Li W. Adams T.E. Huntington J.A. EMBO J. 2006; 25: 2029-2037Crossref PubMed Scopus (151) Google Scholar) and heparin cofactor II (22Baglin T.P. Carrell R.W. Church F.C. Esmon C.T. Huntington J.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11079-11084Crossref PubMed Scopus (185) Google Scholar). These mechanisms fail to explain the effect of GAGs on C1-inh. We determined the structure of N-terminally truncated human C1-inh to understand how heparin and related polyanions alter its activity. Protein Expression and Purification—The cDNA clone of human C1-inh (allele V458M) was kindly provided by Susan Clark Bock (9Bock S.C. Skriver K. Nielsen E. Thøgersen H-C. Wiman B. Donaldson V.H. Eddy R.L. Marrinan J. Radziejewska E. Huber R. Shows T.B. Magnusson S. Biochemistry. 1986; 25: 4292-4301Crossref PubMed Scopus (268) Google Scholar). The EcoRI restriction site was eliminated from the gene. The construct of truncated recombinant C1-inh (rC1-inh) contained six histidines at the N terminus followed by residues Thr97–Ala478 (Fig. 1A). The DNA construct was ligated into pPic9K vector (Invitrogen) between SnaBI and EcoRI sites. The Pichia pastoris GS115 strain (Invitrogen) was transformed with the DraI-digested plasmid, and then His+ MutS clones were isolated. Expression was conducted in a fermentor with a 2-liter vessel (Biostat B; B. Braun). A 40-ml starter culture was grown for 2 days in yeast peptone dextrose medium (Invitrogen). The fermentor was loaded with a 1.5-liter medium of 1% (w/v) yeast extract, 2% (w/v) peptone, 1× yeast nitrogen base (Invitrogen), 0.1 m KH2PO4, 4% (v/v) glycerol and inoculated with the starter. After depletion of batch glycerol, the cells were fed with 50% (v/v) glycerol, and the culture was grown until the wet cell weight reached 200 g liter-1. Recombinant C1-inh production was induced with 25% (v/v) methanol and 40% (w/v) sorbitol for 2–3 days. The pH was kept at 6.0 with NH3, the temperature was kept at 25 °C, and phenylmethylsulfonyl fluoride (0.2 mm final concentration) was added twice daily during the induction period. Phenylmethylsulfonyl fluoride and EDTA were added to the clarified supernatant to a final concentration of 0.5 and 25 mm, respectively, and then stored at -70 °C until use. Nickel affinity purification of recombinant C1-inh was performed (Ni2+-nitrilotriacetic acid Superflow; Qiagen). Cation exchange chromatography followed (Source 15S column; GE Healthcare), using a 10-column volume 0–0.5 m NaCl gradient in the following buffer: 20 mm MOPS, 0.1 mm EDTA, pH 7.0. Active and latent rC1-inh forms were separated at this step. The purified proteins were deglycosylated at 4 °C using endoglycosidase H (New England Biolabs) for 1 week. The deglycosylated protein was purified again by cation exchange as above, followed by gel filtration (Sephadex 75 HiLoad 16/60; GE Healthcare). The purified recombinant C1-inh was concentrated to 10–14 mg ml-1. Protein concentration was estimated from the absorbance of the solution at 280 nm using the extinction coefficient of 0.64 ml mg-1 cm-1, calculated from the protein sequence using the Expasy ProtParam tool. Deglycosylation of Recombinant C1-inh—4.6 μg of glycosylated active recombinant C1-inh (in 10 μl) was used to demonstrate heterogeneity caused by N-glycans. Samples were incubated at 37 °C for 1.5 h, with or without 100 units of endoglycosidase H. β-Mercaptoethanol reduced and nonreduced samples were run on 12.5% SDS-PAGE (Fig. 1B). Activity Test of C1-inh Forms—The reactivity of different C1-inh forms was tested with target proteinase C1s and non-target proteinase trypsin. 0.4 μg of activated C1s (Calbiochem) in 10 μl was used throughout. Bovine pancreatic trypsin (T-1426; Sigma) was dissolved and dialyzed in 1 mm HCl, and then 0.2 μg (in 1 μl) was used in all reactions. Three forms of C1-inh were tested. 2.3 μg of lyophilized plasma C1-inh (Berinert P; ZLB Behring), 2.8 μg of active deglycosylated recombinant C1-inh, and 7.4 μg of latent deglycosylated recombinant C1-inh was made up to 5 μl with buffer containing 140 mm NaCl, 20 mm HEPES, 0.1 mm EDTA, pH 7.4, and used in a reaction. Reaction mixtures were incubated at 37 °C for 1 h and analyzed on 12.5% SDS-PAGE (Fig. 1C). The specific activity of recombinant C1-inh was probed with excess C1s under conditions identical to those described above. ∼9 μg of C1s was reacted with 0.31, 0.63, 1.25, and 2.5 μg of active deglycosylated recombinant C1-inh (in a 10-μl volume). Over 90% of the protein was reactive toward C1s, and ∼70% formed a covalent complex (Fig. S1). Thermal Stability Measurements of C1-inh Forms—Dilutions with 140 mm NaCl, 20 mm HEPES, 0.1 mm EDTA, pH 7.4, buffer were made to yield 0.1–0.2 mg ml-1 C1-inh solutions. Differential scanning calorimetry scans were recorded using VP-differential scanning calorimetry (MicroCal) in the range of 30–125 °C, at a rate of 1 °C min-1, with 15-min preincubation at 30 °C and a 2–4-s data accumulation time. Buffer was used as reference. Melting temperatures were read at peak maximas. The results are summarized in Table 1.TABLE 1Melting temperatures of C1-inh formsC1-inh formActiveLatentCleavedPlasma C1-inh60.0 °CNDaND, not determined115.6 °CrC1-inh (glycosylated)53.8 °CNDaND, not determined114.8 °CrC1-inh (deglycosylated)54.0 °C69.0 °CNDaND, not determineda ND, not determined Open table in a new tab Crystallization and Structure Determination of Latent Recombinant C1-inh—Crystallization trials were set up using the hanging drop method at 20–25 °C, 2–4-μl drops with a protein/reservoir solution ratio of 1:1. Crystals grew in condition 35 (Crystal Screen I; Hampton Research) within 2 weeks. The crystals were cryoprotected by soaking in reservoir solution containing 20% (v/v) glycerol. Collections of diffraction data were carried out at 100 K on beamline X11 of the EMBL outstation at DESY (Hamburg, Germany) (wave-length 0.8162 Å). The asymmetric unit contained one molecule. Data were processed using the XDS package (23Kabsch W. J. Appl. Crystallogr. 1993; 26: 795-800Crossref Scopus (3233) Google Scholar). The structure was solved by molecular replacement using MOLREP of the CCP4 package (24Project Collaborative Computational Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar). Molecular replacement using structures of individual serpins failed, so a superimposed structure ensemble of various serpins was used as a search model. The following structures were acquired from the Protein Data Bank, quoted with respective Protein Data Bank codes: 4CAA, 1QMB, 1E05, 1DVN, 1C8O, 1JTI, 1JJO, 1MTP, 1HLE, and 1JRR. Structurally nonconserved regions, N terminus, and the reactive center loop (RCL) were removed from the search model of molecular replacement using DeepView (25Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9590) Google Scholar). Refinement was carried out with the REFMAC5 program, using TLS refinement and restrained maximum likelihood refinement (26Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. 1997; 53: 240-255Crossref PubMed Scopus (13870) Google Scholar). Manual model building was carried out using the program COOT (27Emsley P. Cowtan K. Acta Crystallogr. Sect. D. 2004; 60: 2126-2132Crossref PubMed Scopus (23389) Google Scholar). Residues of the N and C terminus, three short disordered segments, and side chains of some surface residues were not built in the model due to a lack of electron density. The final model contains residues 101–102, 105–137, 142–293, and 296–476, one covalently bound saccharide, three glycerol, and 70 water molecules. 90.4 and 9.6% of the residues are in the most favored and additional favored regions of the Ramachandran plot, respectively. Data collection and refinement statistics are summarized in Table 2.TABLE 2Data and refinement statisticsParameterValueData collectionSpace groupP65Cell dimensionsa, b, c (Å)98.90, 98.90, 94.68α, β, γ (degrees)90.00, 90.00, 120.00Resolution (Å)aValues are in parentheses for the highest resolution shell20.0-2.35 (2.40-2.35)RmergeaValues are in parentheses for the highest resolution shell0.097 (0.652)I/σIaValues are in parentheses for the highest resolution shell20.29 (3.89)Completeness (%)aValues are in parentheses for the highest resolution shell99.8 (100.0)No. of reflectionsObserved reflectionsaValues are in parentheses for the highest resolution shell319,446 (12,799)Unique reflectionsaValues are in parentheses for the highest resolution shell21,930 (1,341)RefinementResolution (Å)20.0-2.35Rwork/Rfreeb5.1% of the reflections were in a test set for monitoring the refinement process0.174/0.218No. of atomsProtein2,858Saccharide and glycerol32Water70B factorsProtein34.20Saccharide and glycerol69.98Water52.79Root mean square deviationsBond lengths (Å)0.020Bond angles (degrees)1.76a Values are in parentheses for the highest resolution shellb 5.1% of the reflections were in a test set for monitoring the refinement process Open table in a new tab Polymerization Tendency of C1-inh Forms—See supplemental materials and Fig. S2. Heparin Affinity Measurements of C1-inh Forms—Heparin affinity chromatography was performed on a 1-ml column (HiTrap Heparin HP; GE Healthcare) in a buffer containing 20 mm HEPES, 0.1 mm EDTA, pH 7.4. 30–300 μg of C1-inh in a 200-μl volume was injected and washed extensively with buffer, and then a linear salt gradient of 0.0–0.5 m NaCl in a 15-ml volume was applied to elute proteins. Absorbance at 280 nm and conductivity were monitored continuously during the gradient (AöKTApurifier; GE Healthcare) (Fig. 4). Modeling—The structures of the serpins latent α1-proteinase inhibitor (28Im H. Woo M-S. Hwang K.Y Yu M.-H. J. Biol. Chem. 2002; 277: 46347-46354Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) (1IZ2), cleaved protein C inhibitor (29Huntington J.A. Kjellberg M. Stenflo J. Structure. 2003; 11: 205-215Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) (1LQ8), and dimeric antithrombin (30Skinner R. Abrahams J.P. Whisstock J.C. Lesk A.M. Carrell R.W. Wardell M.R. J. Mol. Biol. 1997; 266: 601-609Crossref PubMed Scopus (189) Google Scholar) (1E04) were obtained from the Protein Data Bank. The structures of proteinases fXIa (31Jin L. Pandey P. Babine R.E. Gorga J.C. Seidl K.J. Gelfand E. Weaver D.T. Abdel-Meguid S.S. Strickler J.E. J. Biol. Chem. 2005; 280: 4704-4712Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) (1XX9), C1s (32Gaboriaud C. Rossi V. Bally I. Arlaud G.J. Fontecilla-Camps J.C. EMBO J. 2000; 19: 1755-1765Crossref PubMed Scopus (93) Google Scholar) (1ELV), plasma kallikrein (33Tang J. Yu C.L. Williams S.R. Springman E. Jeffery D. Sprengeler P.A. Estevez A. Sampang J. Shrader W. Spencer J. Young W. McGrath M. Katz B.A. J. Biol. Chem. 2005; 280: 41077-41089Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) (2ANW), MASP-2 (34Harmat V. Gál P. Kardos J. Szilágyi K. Ambrus G. Végh B. Náray-Szabó G. Závodszky P. J. Mol. Biol. 2004; 342: 1533-1546Crossref PubMed Scopus (69) Google Scholar) (1Q3X), and C1r (35Budayova-Spano M. Grabarse W. Thielens N.M. Hillen H. Lacroix M. Schmidt M. Fontecilla-Camps J.C. Arlaud G.J. Gaboriaud C. Structure. 2002; 10: 1509-1519Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) (1MD8) were used. MASP-1 and fXIIa were modeled using SWISS-MODEL (25Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9590) Google Scholar). Glycans were added using the GlyProt server (36Bohne-Lang A. von der Lieth C.-W. Nucleic Acids Res. 2005; 33 (-W219): W214Crossref PubMed Scopus (184) Google Scholar). All proteinases were truncated to the beginning of the activation peptide, so only the catalytic serine proteinase domains were retained. Electrostatics was calculated using APBS (37Baker N.A. Sept D. Joseph S. Holst M.J. McCammon J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10037-10041Crossref PubMed Scopus (5877) Google Scholar) and PDB2PQR. Crystal contacts were evaluated using PISA (38Krissinel E. Henrick K. Berthold M.R. Glen R. Diederichs K. Kohlbacher O. Fischer I. Computational Life Sciences, First International Symposium, CompLife 2005 Konstanz, Germany, September 25–27, 2005 Proceedings. 2005: 163-174Google Scholar). Blind Docking—The structure diagram and the released torsion angles of the docked heparin disaccharide are depicted in Fig. 5C, derived from protein-saccharide complex structures 1T8U and 1XMN. Residue pairs SGN2-IDS3 in Protein Data Bank entry 1T8U and SGN4-IDS5 of chain U in Protein Data Bank entry 1XMN were taken as models of the disaccharide in twist-boat and chair conformations, respectively. Initial calculations with both models yielded similar docked ensembles, so further calculations were carried out with saccharide rings in chair conformation. Calculations were carried out on our C1-inh crystal structure (Protein Data Bank code 2OAY). Active antithrombin both in heparin-bound (39Jin L. Abrahams J.P. Skinner R. Petitou M. Pike R.N. Carrell R.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14683-14688Crossref PubMed Scopus (638) Google Scholar) (1AZX) and -free conformations (1E04) were also used for method validation. The macromolecules were prealigned using AMoRe of the CCP4 suite. Structures were prepared for docking in AutoDockTools; polar hydrogen atoms were added to the structures, and Gasteiger and Kollman united atom charges were used for the ligand and protein atoms, respectively. First docking was carried out on the whole protein represented with coarse grid maps (100 × 96 × 86 grid points with 1.0-Å spacing). The best solution was used for further docking with fine grid maps (108 × 108 × 108 grid points with 0.375-Å spacing). AutoGrid and AutoDock were used for calculation of grid maps and docking, respectively (using the Lamarckian Genetic Algorithm) (40Morris G.M. Goodsell D.S. Halliday R.S. Huey R. Hart W.E. Belew R.K. Olson A.J. J. Comput. Chem. 1998; 19: 1639-1662Crossref Scopus (9210) Google Scholar). Docking was carried out with default parameters. The number of trials was set to 100, with an initial population of 250 individuals, 2 × 107 energy evaluations, and 27,000 generations. The resulting docked structures were clustered with 3- and 1-Å root mean square deviation tolerances for the first and second docking runs. Visualization—Figures were prepared using PyMOL (DeLano Scientific). Additional figures were produced with InkScape, CS ChemDraw Pro (CambridgeSoft), and Origin (MicroCal). Expression and Structure Determination—A C1-inh serpin domain construct (in which the first 96 amino acids of the wild-type protein were replaced with a His-tag) was engineered to reduce heterogeneity and to aid purification. This truncation removes the nonconserved part of C1-inh and leaves most of the biological activities essentially unchanged (11Coutinho M. Aulak K.S. Davis A.E. II I J. Immunol. 1994; 153: 3648-3654PubMed Google Scholar, 12Bos I.G.A. Lubbers Y.T.P. Roem D. Abrahams J.P. Hack C.E. Eldering E. J. Biol. Chem. 2003; 278: 29463-29470Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) (Fig. 1A). The truncated rC1-inh, expressed in P. pastoris, was visualized as a smear between 50 and 100 kDa on SDS-PAGE, since it was heterogeneously glycosylated (Fig. 1B). Purification yielded two forms; one formed a covalent complex with C1s, and the other did not (Fig. 1C). N-Glycans were removed enzymatically, resulting in a homogeneous protein migrating at ∼44 kDa. Differential scanning calorimetry measurement of the inactive form showed a melting temperature between that of the active and cleaved rC1-inh forms (Table 1). The noninhibitory form was crystallized, and a full data set was collected. The structure was determined using molecular replacement and refined to 2.35 Å resolution (Table 2). Overall Structure—Our structure represents the serpin domain (residues 113–478) and a small portion of the N-terminal domain (residues 97–112). The overall structure of rC1-inh resembles those of other serpins (1Gettins P.G.W. Chem. Rev. 2002; 102: 4751-4803Crossref PubMed Scopus (997) Google Scholar), with highly conserved nine α-helices and three β-sheets present (Fig. 2A). The His-tag is not observed, and the remaining part of the unique N-terminal domain is poorly ordered. This small portion of the N-terminal domain is anchored with two nonconserved disulfide bonds along the D and E helices. Structural alignment with other serpin structures reveals major differences at helix D and the loop connecting it with the second strand of sheet A (s2A), where the helix is shortened to 2 turns and s2A is shortened by 2 residues (Fig. 2, A and B). The uncleaved RCL is incorporated into sheet A, forming s4A. Insertion occurred up to position P5, where P1-P1′ denotes the Arg444-Thr445 scissile bond (Fig. 3A). This classifies the crystallized form of rC1-inh as latent (41Mottonen J. Strand A. Symersky J. Sweet R.M. Danley D.E. Geoghegan K.F. Gerard R.D. Goldsmith E.J. Nature. 1992; 355: 270-273Crossref PubMed Scopus (528) Google Scholar). A well defined hydrogen bonding network and burial of reactive center residues explain the loss of inhibitory activity. A striking feature of our structure is that the C-terminal P′ part of the RCL forms the new seventh strand of sheet A, making it a fully anti-parallel β-sheet (Figs. 2A and 3A). This segment forms a flexible surface loop in all other known latent serpin structures (28Im H. Woo M-S. Hwang K.Y Yu M.-H. J. Biol. Chem. 2002; 277: 46347-46354Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 41Mottonen J. Strand A. Symersky J. Sweet R.M. Danley D.E. Geoghegan K.F. Gerard R.D. Goldsmith E.J. Nature. 1992; 355: 270-273Crossref PubMed Scopus (528) Google Scholar, 42Carrell R.W. Stein P.E. Fermi G. Wardell M.R. Structure. 1994; 2: 257-270Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). Apart from backbone strand-strand interactions, hydrogen bonds with the side chains of Gln385 and Gln420 stabilize its conformation. The conformation of the nearby C-terminal tail (encompassing the last 6 residues of the protein) is also unique to rC1-inh. The conformation of this tail is conserved in known serpin structures (1Gettins P.G.W. Chem. Rev. 2002; 102: 4751-4803Crossref PubMed Scopus (997) Google Scholar, 43Irving J.A. Pike R.N. Lesk A.M. Whisstock J.C. Genome Res. 2000; 10: 1845-1864Crossref PubMed Scopus (512) Google Scholar), since the Cα positions of the highly conserved Pro391 of α1-proteinase inhibitor and equivalent prolines in other structures deviate no more than 3 Å (Fig. 3C). In other serpins, the C-terminal tail is placed to the side of strand s6A in sheet A. In contrast, this segment of rC1-inh is folded in a topologically unique way. It is on the other side of the RCL, and the conserved Pro476 is moved away by more than 10 Å (Fig. 3B) compared with the position found in other serpins (Fig. 3C). This conformational change can solely be attributed to a rigid body rotation of backbone around Tyr474, whose Ψ angle made an almost complete turn of ∼180°.FIGURE 3Novel interactions in latent rC1-inh structure. A, stereo view of sheet A of rC1-inh (cyan and magenta strands) and its extended hydrogen bond network stabilizing the entire RCL (magenta). B, stereo view of the C-terminal tail with electron density map contoured at the 1.0 σ level. The tail (green) differs not only in the position but also in the way it is folded around the RCL (magenta). C, stereo view of the C-terminal tail of latentα1-proteinase inhibitor. Orientation and coloring is the same as in B, to make the images comparable. Note that the conserved position of Pro391 is occupied with P7′-P8′ RCL residues in rC1-inh instead of the equivalent Pro476. D, schematic drawing of the hypothetical mechanism of active-latent transition. Latent serpins have their sheet A extended, where the RCL (magenta) inserts in sheet A without RCL cleavage. Strands of sheet C (depicted here as the two-stranded sheet) tilt to allow the RCL to pass, deduced from latent structures known so far (top row). Sheet A is seven-stranded in rC1-inh, since the entire RCL is inserted, forming two new strands (bottom row). This imposes a steric clash with the conserved C terminus (green) if left unaltered. Latency transition is probably enabled through