ACE2 X-Ray Structures Reveal a Large Hinge-bending Motion Important for Inhibitor Binding and Catalysis
2004; Elsevier BV; Volume: 279; Issue: 17 Linguagem: Inglês
10.1074/jbc.m311191200
ISSN1083-351X
AutoresPaul Towler, Bart L. Staker, Sridhar G. Prasad, Saurabh Menon, Jin Tang, Thomas F. Parsons, D. H. Ryan, Martin Fisher, David J. Williams, Natalie A. Dales, Michael A. Patane, Michael W. Pantoliano,
Tópico(s)Receptor Mechanisms and Signaling
ResumoThe angiotensin-converting enzyme (ACE)-related carboxypeptidase, ACE2, is a type I integral membrane protein of 805 amino acids that contains one HEXXH + E zinc-binding consensus sequence. ACE2 has been implicated in the regulation of heart function and also as a functional receptor for the coronavirus that causes the severe acute respiratory syndrome (SARS). To gain further insights into this enzyme, the first crystal structures of the native and inhibitor-bound forms of the ACE2 extracellular domains were solved to 2.2- and 3.0-Å resolution, respectively. Comparison of these structures revealed a large inhibitor-dependent hinge-bending movement of one catalytic subdomain relative to the other (∼16°) that brings important residues into position for catalysis. The potent inhibitor MLN-4760 ((S,S)-2-{1-carboxy-2-[3-(3,5-dichlorobenzyl)-3H-imidazol4-yl]-ethylamino}-4-methylpentanoic acid) makes key binding interactions within the active site and offers insights regarding the action of residues involved in catalysis and substrate specificity. A few active site residue substitutions in ACE2 relative to ACE appear to eliminate the S2′ substrate-binding subsite and account for the observed reactivity change from the peptidyl dipeptidase activity of ACE to the carboxypeptidase activity of ACE2. The angiotensin-converting enzyme (ACE)-related carboxypeptidase, ACE2, is a type I integral membrane protein of 805 amino acids that contains one HEXXH + E zinc-binding consensus sequence. ACE2 has been implicated in the regulation of heart function and also as a functional receptor for the coronavirus that causes the severe acute respiratory syndrome (SARS). To gain further insights into this enzyme, the first crystal structures of the native and inhibitor-bound forms of the ACE2 extracellular domains were solved to 2.2- and 3.0-Å resolution, respectively. Comparison of these structures revealed a large inhibitor-dependent hinge-bending movement of one catalytic subdomain relative to the other (∼16°) that brings important residues into position for catalysis. The potent inhibitor MLN-4760 ((S,S)-2-{1-carboxy-2-[3-(3,5-dichlorobenzyl)-3H-imidazol4-yl]-ethylamino}-4-methylpentanoic acid) makes key binding interactions within the active site and offers insights regarding the action of residues involved in catalysis and substrate specificity. A few active site residue substitutions in ACE2 relative to ACE appear to eliminate the S2′ substrate-binding subsite and account for the observed reactivity change from the peptidyl dipeptidase activity of ACE to the carboxypeptidase activity of ACE2. The angiotensin-converting enzyme (ACE) 1The abbreviations used are: ACE, angiotensin-converting enzyme; sACE, somatic angiotensin-converting enzyme; tACE, testicular or germinal angiotensin-converting enzyme; SARS, severe acute respiratory syndrome; r.m.s., root mean square; Z, benzyloxycarbonyl. -related carboxypeptidase, ACE2, is a type I integral membrane protein of 805 amino acids that contains one HEXXH + E zinc-binding consensus sequence (1Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: E1-E9Crossref PubMed Google Scholar, 2Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1656) Google Scholar). The catalytic domain of ACE2 is 42% identical to that of its closest homolog, somatic angiotensin-converting enzyme (sACE; EC 3.4.15.1), a peptidyl dipeptidase that plays an important role in the renin angiotensin system for blood pressure homeostasis. The loss of ACE2 in knockout mice has no effect on blood pressure, but reveals ACE2 as an essential regulator of heart function (3Crackower M.A. Sarao R. Oudit G.Y. Yagil C. Kozieradzki I. Scanga S.E. Oliveira-dos-Santos A.J. da Costa J. Zhang L. Pei Y. Scholey J. Ferrario C.M. Manoukian A.S. Chappell M.C. Backx P.H. Yagil Y. Penninger J.M. Nature. 2002; 417: 822-828Crossref PubMed Scopus (1412) Google Scholar). In a recent discovery, ACE2 was identified as a functional receptor for the coronavirus that is linked to the severe acute respiratory syndrome (SARS) (4Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzuriaga K. Greenough T.C. Choe H. Farzan M. Nature. 2003; 426: 450-454Crossref PubMed Scopus (4100) Google Scholar, 5Xiao X. Chakraborti S. Dimitrov A.S. Gramatikoff K. Dimitrov D.S. Biochem. Biophys. Res. Commun. 2003; 312: 1159-1164Crossref PubMed Scopus (302) Google Scholar). The physiological differences observed in the phenotypes of ACE (6Krege J.H. John S.W. Langenbach L.L. Hodgin J.B. Hagaman J.R. Bachman E.S. Jennette J.C. O'Brien D.A. Smithies O. Nature. 1995; 375: 146-148Crossref PubMed Scopus (606) Google Scholar, 7Esther Jr., C.R. Howard T.E. Marino E.M. Goddard J.M. Capecchi M.R. Bernstein K.E. Lab. Investig. 1996; 74: 953-965PubMed Google Scholar) and/or ACE2 (3Crackower M.A. Sarao R. Oudit G.Y. Yagil C. Kozieradzki I. Scanga S.E. Oliveira-dos-Santos A.J. da Costa J. Zhang L. Pei Y. Scholey J. Ferrario C.M. Manoukian A.S. Chappell M.C. Backx P.H. Yagil Y. Penninger J.M. Nature. 2002; 417: 822-828Crossref PubMed Scopus (1412) Google Scholar) knockout mice presumably reflect the significant differences in substrate specificity and reactivity between these enzymes. Many substrates for ACE2 were identified by screening biologically active peptides (8Vickers C. Hales P. Kaushik V. Dick L. Gavin J. Tang J. Godbout K. Parsons T. Baronas E. Hsieh F. Acton S. Patane M. Nichols A. Tummino P. J. Biol. Chem. 2002; 277: 14838-14843Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar). In all cases, only carboxypeptidase activity was found. Of the seven best in vitro peptide substrates identified (kcat/Km > 105m-1 s-1), proline and leucine are the preferred P1 residues, with a partiality for hydrophobic residues in the P1′ position, although basic residues at P1′ are also cleaved (peptide-binding subsites in proteins are as previously defined (9Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4766) Google Scholar)). Some of the best in vitro peptide substrates are apelin-13, des-Arg9-bradykinin, angiotensin II, and dynorphin A-(1Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: E1-E9Crossref PubMed Google Scholar, 2Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1656) Google Scholar, 3Crackower M.A. Sarao R. Oudit G.Y. Yagil C. Kozieradzki I. Scanga S.E. Oliveira-dos-Santos A.J. da Costa J. Zhang L. Pei Y. Scholey J. Ferrario C.M. Manoukian A.S. Chappell M.C. Backx P.H. Yagil Y. Penninger J.M. Nature. 2002; 417: 822-828Crossref PubMed Scopus (1412) Google Scholar, 4Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzuriaga K. Greenough T.C. Choe H. Farzan M. Nature. 2003; 426: 450-454Crossref PubMed Scopus (4100) Google Scholar, 5Xiao X. Chakraborti S. Dimitrov A.S. Gramatikoff K. Dimitrov D.S. Biochem. Biophys. Res. Commun. 2003; 312: 1159-1164Crossref PubMed Scopus (302) Google Scholar, 6Krege J.H. John S.W. Langenbach L.L. Hodgin J.B. Hagaman J.R. Bachman E.S. Jennette J.C. O'Brien D.A. Smithies O. Nature. 1995; 375: 146-148Crossref PubMed Scopus (606) Google Scholar, 7Esther Jr., C.R. Howard T.E. Marino E.M. Goddard J.M. Capecchi M.R. Bernstein K.E. Lab. Investig. 1996; 74: 953-965PubMed Google Scholar, 8Vickers C. Hales P. Kaushik V. Dick L. Gavin J. Tang J. Godbout K. Parsons T. Baronas E. Hsieh F. Acton S. Patane M. Nichols A. Tummino P. J. Biol. Chem. 2002; 277: 14838-14843Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar, 9Schechter I. Berger A. Biochem. Biophys. Res. Commun. 1967; 27: 157-162Crossref PubMed Scopus (4766) Google Scholar, 10Turner A.J. Hooper N.M. Trends Pharmacol. Sci. 2002; 23: 177-183Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 11Danilczyk U. Eriksson U. Crackower M.A. Penninger J.M. J. Mol. Med. 2003; 81: 227-234Crossref PubMed Scopus (64) Google Scholar, 12Oudit G.Y. Crackower M.A. Backx P.H. Penninger J.M. Trends Cardiovasc. Med. 2003; 13: 93-101Crossref PubMed Scopus (202) Google Scholar, 13Natesh R. Schwager S.L. Sturrock E.D. Acharya K.R. Nature. 2003; 421: 551-554Crossref PubMed Scopus (686) Google Scholar). The longest peptide substrate identified is a 36-residue peptide, apelin-36 (8Vickers C. Hales P. Kaushik V. Dick L. Gavin J. Tang J. Godbout K. Parsons T. Baronas E. Hsieh F. Acton S. Patane M. Nichols A. Tummino P. J. Biol. Chem. 2002; 277: 14838-14843Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar). An examination of the ACE2 and ACE literature may be found in recently published reviews (10Turner A.J. Hooper N.M. Trends Pharmacol. Sci. 2002; 23: 177-183Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 11Danilczyk U. Eriksson U. Crackower M.A. Penninger J.M. J. Mol. Med. 2003; 81: 227-234Crossref PubMed Scopus (64) Google Scholar, 12Oudit G.Y. Crackower M.A. Backx P.H. Penninger J.M. Trends Cardiovasc. Med. 2003; 13: 93-101Crossref PubMed Scopus (202) Google Scholar). We report here the first crystal structures of the extracellular metallopeptidase domain of ACE2 in its native and inhibitor-bound states and discuss the influence of these structures in understanding the substrate specificity and catalytic mechanism of the enzyme. While preparing these ACE2 structures for publication, two reports on the crystal structures of testicular angiotensin-converting enzyme (tACE) and Drosophila ACE appeared in the literature (13Natesh R. Schwager S.L. Sturrock E.D. Acharya K.R. Nature. 2003; 421: 551-554Crossref PubMed Scopus (686) Google Scholar, 14Kim H.M. Shin D.R. Yoo O.J. Lee H. Lee J.O. FEBS Lett. 2003; 538: 65-70Crossref PubMed Scopus (108) Google Scholar). Protein Expression and Purification—A truncated extracellular form of human ACE2 (residues 1-740) was expressed in baculovirus and purified as described previously (8Vickers C. Hales P. Kaushik V. Dick L. Gavin J. Tang J. Godbout K. Parsons T. Baronas E. Hsieh F. Acton S. Patane M. Nichols A. Tummino P. J. Biol. Chem. 2002; 277: 14838-14843Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar). The signal sequence (residues 1-18) is presumably removed upon secretion from Sf9 cells. The molecular mass of the purified enzyme is 89.6 kDa by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, which is greater than the theoretical molecular mass of 83.5 kDa expected from the sequence (residues 19-740). The difference of ∼6 kDa is believed to be due to glycosylation at the seven predicted N-linked glycosylation sites for this protein. Crystallization—Briefly, 2 μl of purified ACE2 (5 mg/ml) was combined with an equal volume of reservoir solution, and crystals were grown by hanging drop vapor diffusion at 16-18 °C. The best crystallization reservoir solution conditions for native ACE2 were found to be 100 mm Tris-HCl (pH 8.5), 200 mm MgCl2, and 14% polyethylene glycol 8000. Under these conditions, it took ∼2 weeks to grow single crystals suitable for x-ray diffraction. Similarly, diffraction-quality ACE2 crystals were also grown in the presence of an ACE2 inhibitor, MLN-4760 (ML00106791; (S,S)-2-{1-carboxy-2-[3-(3,5-dichlorobenzyl)-3H-imidazol-4-yl]-ethylamino}-4-methylpentanoic acid). Compound MLN-4760 corresponds to compound 16 of Dales et al. (15Dales N.A. Gould A.E. Brown J.A. Calderwood E.F. Guan B. Minor C.A. Gavin J.M. Hales P. Kaushik V.K. Stewart M. Tummino P.J. Vickers C.S. Ocain T.D. Patane M.A. J. Am. Chem. Soc. 2002; 124: 11852-11853Crossref PubMed Scopus (151) Google Scholar). Crystallization trials used 2 μl of reservoir solution plus 2 μl of ACE2 at 5.9 mg/ml containing 0.1 mm inhibitor. The best diffracting ACE2-inhibitor complex crystals were grown in the presence of 19% polyethylene glycol 3000, 100 mm Tris-HCl (pH 7.5), and 600 mm NaCl. Data Collection and Structure Determination—The best data set for native ACE2 was at 2.2-Å resolution and was collected at the Advanced Photon Source (Argonne National Laboratory). A total of 44 x-ray data sets were collected for native ACE2, including a large number of heavy atom soaks of atoms that had good anomalous signals. The data sets for each derivative were collected at different wavelengths to maximize the anomalous signals for the bound heavy atoms. Native ACE2 data were collected to 2.2-Å resolution at λ = 1.28 Å to maximize the anomalous signal at the zinc absorption edge. The heavy atom positions were determined and confirmed by a combination of visual inspection of Patterson maps and automatic search procedures, which included SHAKE 'N BAKE (16Hauptman H.A. Methods Enzymol. 1997; 277: 3-13Crossref PubMed Scopus (36) Google Scholar) and SHELXD (17Abrahams J.P. de Graaff R.A. Curr. Opin. Struct. Biol. 1998; 8: 601-605Crossref PubMed Scopus (18) Google Scholar). The heavy atom parameters were refined and optimized using the computer programs SHARP (18Bricogne G. Vonrhein C. Flensburg C. Schiltz M. Paciorek W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 2023-2030Crossref PubMed Scopus (554) Google Scholar), MLPHARE (19Otwinowski Z. Wolf W. Evans P.R. Leslie A.G.W. Isomorphous Replacement and Anomalous Scattering: Proceedings of the CCP4 Study Weekend. Daresbury Laboratory, Warrington, United Kingdom1991: 80-86Google Scholar), and XHEAVY (20McRee D.E. Practical Protein Crystallography.2nd Ed. Academic Press, Inc., San Diego, CA1999Google Scholar). The experimental phases were improved by solvent flattening and histogram matching. Once the native ACE2 structure was determined, it was used to solve the inhibitor-bound structure of ACE2 to 3.0-Å resolution using molecular replacement methods that employed the program AMoRe in the CCP4 software suite (21Navaza J. Saludjian P. Methods Enzymol. 1997; 276: 581-594Crossref PubMed Scopus (368) Google Scholar). The native structure was split into two subdomains: subdomains I and II (see Fig. 3 for definition). Subdomain II was used for molecular replacement and refined in REFMAC5, which resulted in the appearance of electron density for subdomain I. Subdomain I was then fitted into the density by hand, and the structure was refined as a whole. Final refinement was accomplished using the software suite CNX (22Brunger 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. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16967) Google Scholar). Native ACE2 Structure—The three-dimensional structure of the extracellular region of native ACE2 was determined by multiple isomorphous replacement with anomalous scattering and refined to a crystallographic R-factor of 23.5% (Rfree = 28.7%) at 2.2-Å resolution. The heavy atom data statistics are summarized in Table I, and the refinement statistics for native ACE2 are summarized in Table II.Table IHeavy atom data statistics for human native ACE2DerivativeNative (Zu)pCMBHgCl2PIPK2PtCl4Heavy atomZnHgHgPtPtMolarity (mm)NA1111Length of soak (day(s))NA3.530130No. sites/asymmetric unitaEach asymmetric unit contains one human ACE2 protein.11122Wavelength (Å)bData were collected at the Brookhaven National Laboratory (NSLS, beam line X25) or at the Argonne National Laboratory (APS, beam line sector 32, COM-CAT). The wavelength for the native (zinc) data set was 1.2824 Å to maximize the anomalous signal at the zinc absorption edge.1.28241.0091.0091.0721.072Unique reflections49,286cValues do not include Bijvoet pairs. Inclusion of Bijvoet pairs increases the number of reflections to 91,550 for native (zinc) ACE2 and 41,716 for the p-chloromercuribenzoate derivative.21,652cValues do not include Bijvoet pairs. Inclusion of Bijvoet pairs increases the number of reflections to 91,550 for native (zinc) ACE2 and 41,716 for the p-chloromercuribenzoate derivative.17,42113,15214,087Resolution (Å)40-2.230-2.930-3.030-3.430-3.3Completeness (%)96.396.690.695.494.2Rsym (%)dRsym=∑|Ii-Im|/∑Im, where Ii is the intensity of the measured reflection and Im is the mean intensity of all symmetry-related reflections.5.710.510.49.711.6RmergeeRmerge=Σ|FPH-FP|/Σ|FPH|.NA21.337.620.621.8RcullisfRcullis=Σ|(FPH±FP)-FH(calc)|/Σ|FPH-FP|.0.940.730.930.960.97Phasing powergPhasing power = FH/ERMS, where ERMS is the residual lack of closure.1.571.510.660.450.39a Each asymmetric unit contains one human ACE2 protein.b Data were collected at the Brookhaven National Laboratory (NSLS, beam line X25) or at the Argonne National Laboratory (APS, beam line sector 32, COM-CAT). The wavelength for the native (zinc) data set was 1.2824 Å to maximize the anomalous signal at the zinc absorption edge.c Values do not include Bijvoet pairs. Inclusion of Bijvoet pairs increases the number of reflections to 91,550 for native (zinc) ACE2 and 41,716 for the p-chloromercuribenzoate derivative.d Rsym=∑|Ii-Im|/∑Im, where Ii is the intensity of the measured reflection and Im is the mean intensity of all symmetry-related reflections.e Rmerge=Σ|FPH-FP|/Σ|FPH|.f Rcullis=Σ|(FPH±FP)-FH(calc)|/Σ|FPH-FP|.g Phasing power = FH/ERMS, where ERMS is the residual lack of closure. Open table in a new tab Table IIRefinement statistics for native and inhibitor-bound ACE2 structuresNative ACE2 (PDB code 1R42)Inhibitor-bound ACE2 (PDB code 1R4L)Resolution (Å)46.7-2.20 (2.34-2.20)43.3-3.0 (3.19-3.00)No. reflections47,465 (5982)17,228 (2250)Rsym (%)aSee Footnote d of Table I.5.7 (40.8)7.0 (20.4)Completeness (%)96.3 (81.8)96.8 (85.1)Space groupC2C2a103.64100.53b89.4686.51c112.40105.86β109.15103.65Unit cell volume (Ås)986,854894,383Solvent content (%)bVsolvent = 1-1.23/Vm, where Vm is the volume of protein in the unit cell/volume of unit cell, assuming one molecule/asymmetric unit and four asymmetric units in the monoclinic unit cell.5353Molecules/asymmetric unit11Reflections used in Rfree47981723No. protein atoms51655147No. solvent atoms30213No. zinc atoms11No. chloride atoms11No. sugar atoms4228R-factor (%)23.5 (37.9)25.3 (37.9)Rfree (%)28.7 (39.8)33.7 (46.0)r.m.s. deviations from ideal stereochemistryBond lengths (Å)0.0080.008Bond angles1.4°1.5°Dihedrals21.7°22.2°Impropers0.92°0.97°Mean B-factor (all atoms; Å2)59.974.5a See Footnote d of Table I.b Vsolvent = 1-1.23/Vm, where Vm is the volume of protein in the unit cell/volume of unit cell, assuming one molecule/asymmetric unit and four asymmetric units in the monoclinic unit cell. Open table in a new tab The extracellular region of the human ACE2 enzyme is composed of two domains. The first is a zinc metallopeptidase domain (residues 19-611), which is ∼42% identical to the corresponding domains of human sACE and tACE (Fig. 1). Electron density near the active site of native ACE2 is shown in Fig. 2a. An α-carbon trace for this metallopeptidase domain of ACE2 is shown in Fig. 3A. The second domain is located at the C terminus (residues 612-740) and is ∼48% identical to human collectrin (23Zhang H. Wada J. Hida K. Tsuchiyama Y. Hiragushi K. Shikata K. Wang H. Lin S. Kanwar Y.S. Makino H. J. Biol. Chem. 2001; 276: 17132-17139Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Unfortunately, the electron density map for much of the collectrin homology domain is weak. Only half of this domain is visible in the electron density map, and what can be seen is ambiguous due to topology and connectivity issues.Fig. 2Experimental electron density maps for native and inhibitor-bound ACE2 structures. a,an |Fo|-|Fc| omit electron density map of the zinc-binding site of the native ACE2 structure (His374-His378, His401-Glu406), calculated with phases from the refined model at 2.2-Å resolution. The map is contoured at 3σ. b, an |Fo|-|Fc| omit electron density map of MLN-4760, zinc, and the three metal-binding ligands of the protein (His374, His378, and Glu402), calculated with phases from the refined model at 3.0-Å resolution. The map is contoured at 3σ.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The metallopeptidase domain of ACE2 can be further divided into two subdomains (I and II) (Fig. 3B), which form the two sides of a long and deep cleft with dimensions of ∼40 Å long by ∼15 Å wide by ∼25 Å deep. The two catalytic subdomains are connected only at the floor of the active site cleft. One prominent α-helix (helix 17, residues 511-531) connects the two subdomains and forms part of the floor of the canyon. The deeply recessed and shielded proteolytic active site of ACE2 is a common structural feature of proteases and exists to avoid hydrolysis of correctly folded and functional proteins (24Fulop V. Bocskei Z. Polgar L. Cell. 1998; 94: 161-170Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 25Rockel B. Peters J. Kuhlmorgen B. Glaeser R.M. Baumeister W. EMBO J. 2002; 21: 5979-5984Crossref PubMed Scopus (33) Google Scholar) and is also consistent with the profiles of binding of tethered inhibitors to sACE (26Pantoliano M.W. Holmquist B. Riordan J.F. Biochemistry. 1984; 23: 1037-1042Crossref PubMed Scopus (78) Google Scholar, 27Bernstein K.E. Welsh S.L. Inman J.K. Biochem. Biophys. Res. Commun. 1990; 167: 310-316Crossref PubMed Scopus (24) Google Scholar). The N terminus-containing subdomain I and the C terminus-containing subdomain II are defined in Fig. 3B. This subdomain definition is based on the subdomain movement that was observed upon inhibitor binding (see below). The secondary structure of the metallopeptidase domain of ACE2 is composed of 20 α-helical segments and nine 310 helical segments that together make up ∼62% of the structure (Figs. 1 and 3). This contrasts with just six short β-structural segments that make up ∼3.5% of the structure. Glycosylation is suggested by the presence of electron density at all six potential N-linked sites: Asn53, Asn90, Asn103, Asn322, Asn432, and Asn546 (2Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1656) Google Scholar). The highest density was observed at Asn90, Asn103, and Asn546 and allowed the building of three N-acetylglucosamine groups. Three disulfide bonds of ACE2 (Cys133-Cys141, Cys344-Cys361, and Cys530-Cys542) are conserved in sACE and tACE (Fig. 1). The zinc-binding site is located near the bottom and on one side of the large active site cleft (subdomain I side), nearly midway along its length. The zinc is coordinated by His374, His378, Glu402, and one water molecule (in the native structure). These residues at the zinc-binding site of ACE2 make up the HEXXH + E motif conserved in the zinc metallopeptidase clan MA (28Rawlings N.D. Barrett A.J. Methods Enzymol. 1995; 248: 183-228Crossref PubMed Scopus (696) Google Scholar). A chloride ion (Cl-) is bound in native ACE2, coordinated by Arg169, Trp477, and Lys481 in subdomain II. The larger electron density compared with water supports the assignment of this density to a Cl- ion, as does as the larger than expected distances between the coordinating side chains and the Cl- ion (compared with water H-bonds): N-ϵ and N-η1 of Arg169 are both 3.2 Å from Cl-, indole ring nitrogen of Trp477 is 3.5 Å from Cl-, and N-ϵ of Lys481 is 5.0 Å from Cl-. Inhibitor-bound ACE2 Structure—The structure of ACE2 with an inhibitor bound at the active site was solved by molecular replacement to a resolution of 3.0 Å using the native ACE2 structure. Refinement statistics for the inhibitor-bound ACE2 structure are shown in Table II. The bound compound MLN-4760 potently inhibits human ACE2 (IC50 = 0.44 nm), but weakly inhibits tACE (IC50 > 100 μm) and carboxypeptidase A (IC50 = 27 μm) (15Dales N.A. Gould A.E. Brown J.A. Calderwood E.F. Guan B. Minor C.A. Gavin J.M. Hales P. Kaushik V.K. Stewart M. Tummino P.J. Vickers C.S. Ocain T.D. Patane M.A. J. Am. Chem. Soc. 2002; 124: 11852-11853Crossref PubMed Scopus (151) Google Scholar). The structure of the bound inhibitor is shown in Fig. 2b along with the experimental electron density map near the active site. Ligand-dependent Subdomain Hinge-bending Movement—There is a clear difference between the native and inhibitor-bound ACE2 structures with respect to the distance separating the two subdomains (Fig. 4A). These two subdomains undergo a large inhibitor-dependent hinge-bending movement of one catalytic subdomain relative to the other (∼16°) that causes the deep open cleft in the native form of the enzyme to close around the inhibitor. This movement can be viewed when subdomain II from the native and inhibitor-bound ACE2 structures are superimposed (root mean square (r.m.s.) deviation of 1.41 Å for 409 residues) as shown in Fig. 4A. In this view, subdomain II remains essentially unchanged, but subdomain I moves to close the gap, essentially mimicking the action of a closing clam shell. The α-carbon atoms of some residues near the outer edge of the subdomain gap move as much as ∼13 Å, whereas residues lying near or on the hinge axis (residues 99 and 100, 284-293, 396 and 397, 409 and 410, 433 and 434, 539-548, and 564-568) are nearly stationary. The movement of residues within the active site is shown in Fig. 4B. This view is a close-up of Fig. 4A with subdomain II of the native and inhibitor-bound ACE2 structures superimposed. Many of these residues move as much as 6-9 Å after binding of the inhibitor. Similar subdomain hinge-bending motions have also been observed for other zinc metalloproteases (29Holland D.R. Tronrud D.E. Pley H.W. Flaherty K.M. Stark W. Jansonius J.N. McKay D.B. Matthews B.W. Biochemistry. 1992; 31: 11310-11316Crossref PubMed Scopus (123) Google Scholar, 30Grams F. Dive V. Yiotakis A. Yiallouros I. Vassiliou S. Zwilling R. Bode W. Stocker W. Nat. Struct. Biol. 1996; 3: 671-675Crossref PubMed Scopus (149) Google Scholar). The largest previously observed ligand-dependent movement for metalloproteases is a 14° hinge-bending subdomain motion demonstrated for Pseudomonas aeruginosa elastase (open versus closed) that resulted in an ∼2-Å movement to close the N-terminal/C-terminal subdomain gap (29Holland D.R. Tronrud D.E. Pley H.W. Flaherty K.M. Stark W. Jansonius J.N. McKay D.B. Matthews B.W. Biochemistry. 1992; 31: 11310-11316Crossref PubMed Scopus (123) Google Scholar). Domain closure movements in proteins are a common mechanism for the positioning of critical groups around substrates and inhibitors (31Gerstein M. Lesk A.M. Chothia C. Biochemistry. 1994; 33: 6739-6749Crossref PubMed Scopus (707) Google Scholar, 32Gerstein M. Krebs W. Nucleic Acids Res. 1998; 26: 4280-4290Crossref PubMed Scopus (313) Google Scholar, 33Teague S.J. Nat. Rev. Drug Discov. 2003; 2: 527-541Crossref PubMed Scopus (632) Google Scholar) and also for the trapping of substrate and reaction intermediates (34Knowles J.R. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 1991; 332: 115-121Crossref PubMed Scopus (67) Google Scholar). Inhibitor Binding Interactions and Implications for Substrate Specificity and Catalysis—Both metallopeptidase subdomains of ACE2 are nearly equally involved in binding of the inhibitor MLN-4760. Inspection of the interactions between MLN-4760 and ACE2 revealed important residues responsible for inhibitor binding and presumably for substrate binding and catalysis (Fig. 5). The inhibitor MLN-4760 has two carboxylate groups, one of which binds to the zinc atom by displacing the bound water molecule present in the native ACE2 structure. The zinc coordination sphere (His374, His378, and Glu402) is a subset of the 21 residues of ACE2 that are located within 4.5 Å of the bound inhibitor and that make up the greater part of the active site. Six of the most important of these residues contribute specific H-bonding interactions with MLN-4760 (Fig. 5). The side chains of Arg273, His505, and His345 are H-bonded to the terminal carboxylate of the inhibitor. The carbonyl oxygen atom of Pro346 and the N-ϵ atom of His345 are within H-bonding distance of the secondary amine group of the inhibitor. Thr371 is within H-bonding distance of the imidazole ring of the 3,5-dichlorobenzylimidazole group of MLN-4760. The phenolic group of Tyr515 donates an H-bond to the zinc-bound carboxylate group of the inhibitor. The carboxyl group of Glu375 is within H-bonding distance of the other oxygen atom of the zinc-bound carboxylate of MLN-4760, but is presumably not protonated until peptide hydrolysis occurs (see below). The zinc-bound carboxylate of MLN-4760 appears to mimic the zinc-bound tetrahedral intermediate characteristic of nucleophilic attack of the scissile bond by the zinc-bound water during peptide hydrolysis (35Matthews B.W. Acc. Chem. Res. 1988; 21: 333-340Crossref Scopus (672) Google Scholar). This transition state structure is usually stabilized by H-bonds donated by imidazole, phenolic, or guanidino functional groups of neighboring amino acid side chains in other zinc metalloproteases (30Grams F. Dive V. Yiotakis A. Yiallouros I. Vassiliou S. Zwilling R. Bode W. Stocker W. Nat. Struct. Biol. 1996; 3: 671-675Crossref PubMed Scopus (149) Google Scholar, 35Matthews B.W. Acc. Chem. Res. 1988; 21: 333-340Crossref Scopus (672) Google Scholar). For ACE2, this carboxyl anion stabilization most likely occurs through the phenolic group
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