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. 2004 Apr 23;279(17):17996-8007.
doi: 10.1074/jbc.M311191200. Epub 2004 Jan 30.

ACE2 X-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis

Paul Towler  1 , Bart Staker, Sridhar G Prasad, Saurabh Menon, Jin Tang, Thomas Parsons, Dominic Ryan, Martin Fisher, David Williams, Natalie A Dales, Michael A Patane, Michael W Pantoliano
Affiliations

ACE2 X-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis

Paul Towler et al. J Biol Chem. .

Abstract

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-A resolution, respectively. Comparison of these structures revealed a large inhibitor-dependent hinge-bending movement of one catalytic subdomain relative to the other ( approximately 16 degrees ) 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 S(2)' substrate-binding subsite and account for the observed reactivity change from the peptidyl dipeptidase activity of ACE to the carboxypeptidase activity of ACE2.

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Figures

F<sc>ig</sc>. 3
Fig. 3
Overview of the native ACE2 crystal structure.A, α-carbon trace of the native ACE2 structure looking down into the metallopeptidase active site cleft. The metallopeptidase catalytic domain is colored red. The active site zinc ion is shown as a yellow sphere, and the single bound chloride ion is shown as a green sphere. The S1′ subsite for inhibitor and substrate binding is to the right of the zinc ion, and the S1 subsite is to the left. The collectrin homology domain at the C terminus is disordered and denoted by the green dotted line. B, ribbon diagram of native ACE2 showing the secondary structure and also the two subdomains (I and II) that form the two sides of the active site cleft. The two subdomains are defined as follows: the N terminus- and zinc-containing subdomain I (red), composed of residues 19-102, 290-397, and 417-430; and the C terminus-containing subdomain II (blue), composed of residues 103-289, 398-416, and 431-615. This definition is based on motion observed upon inhibitor binding (see Fig. 4). Zinc and chloride ions are denoted as described for A.
F<sc>ig</sc>. 1
Fig. 1
Sequence alignment of the metallopeptidase domains of human ACE2, sACE, and tACE. The sequences of the catalytic domain of tACE and the C-terminal catalytic domain of sACE are identical (48, 49). The human sACE and ACE2 sequences were obtained from the GenBank™/EBI Data Bank (accession numbers P12821 and AAF99721, respectively). The ClustalW Alignment Tool was used for these sequence alignments (50). The secondary structural elements for native ACE2 were assigned using STRIDE software (51) and are denoted as follows: α-helical segments,- - ▸; 310 helical elements, - - >; and β-structural segments, - - •. These secondary structural elements are color-coded red for subdomain I and blue for subdomain II (subdomains are defined in Fig. 3). Identical residues for all three enzymes are colored red. The zinc-binding residues in all three enzymes are shown in green, and the chloride ion-binding residues in all enzymes are shown in orange. Residues in ACE2 within H-bonding distance of the inhibitor MLN-4760 or in tACE within H-bonding distance of lisinopril are colored blue (13). The Cys residues conserved between ACE2 and ACE are colored magenta. The six predicted N-linked glycosylation sites for the metallopeptidase region of ACE2 are shown (gray N). The beginning of the collectrin homology domain (23) in ACE2 is indicated (▾).
F<sc>ig</sc>. 2
Fig. 2
Experimental 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.
F<sc>ig</sc>. 4
Fig. 4
Superposition of the native and inhibitor-bound ACE2 structures.A, the 409 α-carbon atoms corresponding to subdomain II of the native and inhibitor-bound ACE2 structures were superimposed with an r.m.s. deviation of 1.41 Å. Native ACE2 is colored red, and inhibitor-bound ACE2 is colored green. The zinc ion is shown as a yellow sphere, and the inhibitor MLN-4760 is shown in a ball-and-stick rendering with default atom coloring: gray, carbon; blue, nitrogen; red, oxygen; green, chlorine. This view is looking down the length of the active site cleft and is rotated 90° from that shown in Fig. 3. This perspective illustrates the ∼16° hinge-bending movement of subdomain I relative to subdomain II that occurs upon inhibitor binding to ACE2. B, shown is a close-up view of the active sites of the superimposed native (red) and inhibitor-bound (green) ACE2 structures. This is the same superposition of subdomain II for both structures as described for A. In this perspective, the residues of subdomain I within the active site are shown to move upon inhibitor binding relative to those in subdomain II. The inhibitor MLN-4760 is shown in stick rendering with the same atom color code as described for A. The average movement for residues near the active site is 6-9 Å. The yellow spheres are the two positions of the zinc atom in the native and inhibitor-bound structures. This figure was prepared using MOE 2003.02 software (Chemical Computing Group, Inc.).
F<sc>ig</sc>. 5
Fig. 5
Binding interactions of the inhibitor MLN-4760 at the active site of ACE2.A, the residues of ACE2 that contribute direct binding interactions with the inhibitor MLN-4760 are shown. MLN-4760 is shown in stick rendering with the same atom color code as described in the legend to Fig. 4A, except carbon is orange. The α-helix 11 segment derived from subdomain I has the α-carbon wire colored red, and turns and β-elements derived from subdomain II have the α-carbon wire colored blue. Probable H-bonding interactions are shown as black dashed lines. The zinc ion is shown as a yellow sphere. ACE2 residues coordinating the zinc ion are shown in stick rendering. B, shown is a schematic view of MLN-4760 binding interactions. MLN-4760 is shown in black. Residues derived from subdomain I are red, and residues derived from subdomain II are blue. The equivalent residues in tACE are in given in parentheses. Distances are measured in angstroms.
F<sc>ig</sc>. 6
Fig. 6
Superposition of the ACE2 and tACE structures.A, the α-carbon atoms in lisinopril-bound tACE (13) were superimposed onto the equivalent atoms in inhibitor-bound ACE2 (588 residues) with an r.m.s. deviation of 1.80 Å. MLN-4760-bound ACE2 is magenta, and lisinopril-bound tACE is green. MLN-4760 is shown bound to ACE2 with the same color code described in the legend to Fig. 4A. Similarly, the zinc and chloride ions are shown as described in the legend to Fig. 3. The orientation is the same as that shown for native ACE2 in Fig. 3. Structures were superimposed using MOE 2003.02 software. B, the 21 α-carbon atoms at the inhibitor-bound active site of ACE2 (residues 4.5 Å from the inhibitor) were superimposed onto the equivalent atoms of lisinopril-bound tACE (Protein Data Bank code 1O86) with an r.m.s. deviation of 0.53 Å. The active site of ACE2 and MLN-4760 are shown in default colors, with the inhibitor displayed in stick rendering. Labels are for ACE2 residues only. The active site residues of tACE are shown in orange, with the inhibitor lisinopril colored purple in stick rendering. The zinc ion is shown as a yellow sphere, and the second chloride ion of tACE (CL2) is shown as an orange sphere. This chloride ion site does not exist in ACE2 due to the Glu398 substitution for Pro407 (see “Results and Discussion”). Other important differences between ACE2 and tACE are as follows: Arg273versus Gln281, Phe274versus Thr282, and Tyr510versus Val518, respectively.
F<sc>ig</sc>. 7
Fig. 7
Structural homology for the ACE2 catalytic motif to other members of the HEXXH metallopeptidase clans.A, shown is the structure-based sequence alignment of ACE2, tACE, thermolysin, and neurolysin. The conserved residues correspond to the catalytic motif for these enzymes (colored red (zinc binding) and magenta). Sequence numbering is for ACE2. B, the catalytic motifs for thermolysin and astacin bound to transition state analogs Z-Pheψ(PO2NH)-Leu-Ala and Z-Pro-Lys-Pheψ(PO2CH2)-dl-Ala-Pro-OMe, respectively (30, 42), are compared with the ACE2 complex with MLN-4760. Distances are measured in angstroms. C, shown is the superposition of the catalytic motifs of ACE2 (red) and thermolysin (green). Eight α-carbon atoms corresponding to residues 345, 346, 374-378, and 402 of MLN-4760 bound ACE2 were superimposed onto the equivalent α-carbon atoms of Z-Pro-Lys-Pheψ(PO2CH2)-dl-Ala-Pro-OMe-bound thermolysin (see sequence alignment in A) with an r.m.s. deviation of 0.49 Å. Bound inhibitors are shown in stick rendering with default atom coloring for MLN-4760 and green coloring for Z-Pro-Lys-Pheψ(PO2CH2)-dl-Ala-Pro-OMe. The zinc ion is shown as a yellow sphere. ACE2 labels are black, and thermolysin labels are blue. ψ indicates replacement of the peptide bond by the group in parentheses.
F<sc>ig</sc>. 8
Fig. 8
Proposed mechanism for ACE2-catalyzed hydrolysis of peptide substrates.A, shown is the ES complex and progression to the tetrahedral intermediate. Substrate binding to one subdomain induces a subdomain hinge-bending movement (indicated by large arrows) to close the active site cleft and to bring important residues into position for catalysis. This movement is followed by attack of a zinc-bound water at the carbonyl group of the scissile amide bond to form a tetrahedral intermediate, resulting in transfer of a proton from the attacking water to Glu375 (35). Simultaneously, a proton is transferred from His505 to the leaving nitrogen atom of the P1′ residue. This sp3 hybridized nitrogen is stabilized by H-bonds from Pro346, His505, and/or His345. B, collapse of the tetrahedral intermediate to the products occurs by breaking of the amide C-N bond together with abstraction of a proton from Glu375 by the emerging free nitrogen of the product amino acid. The new emerging product carboxyl group can then transfer a proton back to His505 either directly by exchange between carboxyl oxygen atoms or by exchange with solvent.
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