PDBsum entry 1r4l

Hydrolase

PDB id: 1r4l

Contents

Protein chains

597 a.a. *

20 a.a. *

18 a.a. *

14 a.a. *

Ligands

UNK-UNK-UNK-UNK-UNK-UNK

NAG ×2

XX5

Metals

_CL

_ZN

Waters ×13

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Ace2 X-Ray structures reveal a large hinge-Bending motion important for inhibitor binding and catalysis.

Authors

P.Towler, B.Staker, S.G.Prasad, S.Menon, J.Tang, T.Parsons, D.Ryan, M.Fisher, D.Williams, N.A.Dales, M.A.Patane, M.W.Pantoliano.

Ref.

J Biol Chem, 2004, 279, 17996-18007. [DOI no: 10.1074/jbc.M311191200]

PubMed id

14754895

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.

Figure 4.
FIG. 4. Superposition of the native and inhibitor-bound ACE2 structures. A, the 409 -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.).

Figure 6.
FIG. 6. Superposition of the ACE2 and tACE structures. A, the -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 -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 [PDB] ) 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: Arg273 versus Gln281, Phe^274 versus Thr282, and Tyr510 versus Val518, respectively.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 17996-18007) copyright 2004.

Secondary reference #1

Title

Substrate-Based design of the first class of angiotensin-Converting enzyme-Related carboxypeptidase (ace2) inhibitors.

Authors

N.A.Dales, A.E.Gould, J.A.Brown, E.F.Calderwood, B.Guan, C.A.Minor, J.M.Gavin, P.Hales, V.K.Kaushik, M.Stewart, P.J.Tummino, C.S.Vickers, T.D.Ocain, M.A.Patane.

Ref.

J Am Chem Soc, 2002, 124, 11852-11853. [DOI no: 10.1021/ja0277226]

PubMed id

12358520

Secondary reference #2

Title

Hydrolysis of biological peptides by human angiotensin-Converting enzyme-Related carboxypeptidase.

Authors

C.Vickers, P.Hales, V.Kaushik, L.Dick, J.Gavin, J.Tang, K.Godbout, T.Parsons, E.Baronas, F.Hsieh, S.Acton, M.Patane, A.Nichols, P.Tummino.

Ref.

J Biol Chem, 2002, 277, 14838-14843. [DOI no: 10.1074/jbc.M200581200]

PubMed id

11815627

Figure 2.
Fig. 2. pH dependence of ACE2 proteolytic activity. ACE2-catalyzed hydrolysis reactions were performed with 0.15 nM ACE2 and 50 µM Mca-APK(Dnp) in 1.0 M NaCl, 10 µM ZnCl[2], 0.01% Brij-35, and 50 mM buffer as described under "Experimental Procedures." Rates of hydrolysis of the internally quenched fluorescent peptide Mca-APK(Dnp) were determined by measuring the slope of increase in fluorescence (excitation = 320 nm, emission = 405 nm) under initial velocity conditions ( 10% hydrolysis) over 15-60 min. All values are an average (n = 2), and the S.D. is shown. , sodium acetate; , MES; , bis-Tris propane; circle , CHES; , CAPS.

Figure 3.
Fig. 3. Dependence of ACE2 proteolytic activity on anion concentration. ACE2-catalyzed hydrolysis reactions were performed with 0.15 nM ACE2 and 50 µM Mca-APK(Dnp) in salt, 10 µM ZnCl[2], 0.01% Brij-35, and 50 mM MES, pH 6.5, as described under "Experimental Procedures." Rates of hydrolysis of the internally quenched fluorescent peptide Mca-APK(Dnp) were determined by measuring the slope of increase in fluorescence (excitation = 320 nm, emission = 405 nm) under initial velocity conditions ( 10% hydrolysis) over 15-60 min. All values are an average (n = 2), and the S.D. is shown. , NaCl; , NaBr; , NaF; circle , KCl.

The above figures are reproduced from the cited reference with permission from the ASBMB