Crystal Structure of the N Domain of Human Somatic Angiotensin I-converting Enzyme Provides a Structural Basis for Domain-specific Inhibitor Design

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Human somatic angiotensin I-converting enzyme (sACE) is a key regulator of blood pressure and an important drug target for combating cardiovascular and renal disease. sACE comprises two homologous metallopeptidase domains, N and C, joined by an inter-domain linker. Both domains are capable of cleaving the two hemoregulatory peptides angiotensin I and bradykinin, but differ in their affinities for a range of other substrates and inhibitors. Previously we determined the structure of testis ACE (C domain); here we present the crystal structure of the N domain of sACE (both in the presence and absence of the antihypertensive drug lisinopril) in order to aid the understanding of how these two domains differ in specificity and function. In addition, the structure of most of the inter-domain linker allows us to propose relative domain positions for sACE that may contribute to the domain cooperativity. The structure now provides a platform for the design of “domain-specific” second-generation ACE inhibitors.

Introduction

Angiotensin I-converting enzyme (ACE, EC 3.4.15.1) is a zinc-dependent dipeptidyl carboxypeptidase with diverse physiological functions, including principally that of blood pressure regulation via angiotensin II production and bradykinin inactivation. Somatic ACE (sACE), a type I transmembrane protein, is composed of two homologous catalytic domains (N and C domains), arising from a gene duplication event1 (Figure 1(a)). Recent studies have highlighted the unique physiological roles of the N and C domains as well as evidence for negative cooperativity between them.2, 3, 4 The germinal form of ACE (testis ACE)5 originates from the same gene as somatic ACE, but has a tissue-specific promoter located within intron 12. Testis ACE (tACE) plays a crucial role in reproduction,6 although the nature of its role is still under investigation.7, 8, 9

Enzymatically active forms of the N domain have been found in human ileal fluid10 and in the urine of mild hypertensive patients where it could have an important role in the development of hypertension.11 Despite sharing ∼60% sequence identity with the C domain, the N domain has its own distinctive physicochemical and functional properties. It is thermally more stable than the C domain,12 more resistant to proteolysis under denaturing conditions13 and is less dependent on chloride activation as compared with the C domain.14, 15 Both domains are heavily glycosylated, a feature that has hampered the 3D structure determination of the protein.

The N domain has ten N-linked glycosylation sites of which seven are unique to the N domain. The different glycan profile of the N domain is likely to be responsible for the carbohydrate-mediated dimerisation of the somatic form, which has been described under certain conditions.16, 17 Moreover, the difference in glycosylation could impact on the structural basis for epitope recognition. Indeed, epitope mapping of the N domain has revealed a region that might play a role in the relatively inefficient ectodomain shedding of sACE compared to its germinal isoform.18 Substrates such as the hemoregulatory peptide AcSDKP,19 angiotensin 1–7,20 and the enkephalin precursor Met5-Enk-Arg6-Phe721 are specific for the N domain, whereas the physiological substrates bradykinin and angiotensin I are hydrolysed with similar catalytic efficiency as the C domain. Interestingly, the N domain preferentially hydrolyses the Aβ peptide of the amyloid precursor protein resulting in inhibition of Aβ aggregation and cytotoxicity in cell-based assays,22 although this is not necessarily the case in vivo.23 The widely used ACE inhibitor captopril shows modest selectivity for the N domain.14 Also, the phosphinic peptide inhibitor RXP-407 has a dissociation constant three orders of magnitude lower for the N domain of the enzyme.24

Despite the physiological importance of ACE and the fact that it is a well-validated therapeutic target, the first 3D structures were only determined almost half a century after its discovery.25, 26, 27 The crystal structures of tACE and the Drosophila homologue AnCE reveal a predominantly α-helical structure with two internal chambers linked by a constriction where the active-site zinc is coordinated by the two histidine residues of the HEXXH motif and a single downstream glutamate. Comparison of the ACE structure with that of the ACE 2 homologue,28, 29 which more closely resembles the N domain, provides a molecular basis for the different mechanisms involved in the dipeptidase and carboxypeptidase activities of these two metalloenzymes.

We have now determined the crystal structures of both native N domain of sACE and N domain complexed with the inhibitor lisinopril at a resolution of 3.0 Å (Table 1). The structure, including that of the inter-domain linker region reveals differences in the active site and chloride binding. The well ordered linker region enables us to speculate on the relative domain orientations in somatic ACE, which provides some insight into the basis for domain cooperativity.

Section snippets

Overall structure of the N domain

The N domain was crystallised in the space group C2221 with two molecules per asymmetric unit. The overall fold consists of a mainly helical secondary structure, with the same topology as tACE (Figure 1(b)). Briefly, the N domain has an ellipsoid shape with a central groove dividing it into two sub-domains, one of which contains the N-terminal region that covers the central binding cavity (Figure 1(b) and (e)). There are 27 helices, of which 18 are α-helices, five are short 310 helices and four

Conclusion

The determination of the N domain structure is an important advance in the understanding of the overall structure of human sACE. It allows insight into the specificity and the physiological significance of the N and C domain active sites. There has been a renewed interest in the molecular basis for the enzyme activity of ACE, a cardiovascular drug target of enormous clinical importance. The structural details of previously determined tACE (C domain of sACE) along with the N domain of sACE

Expression and purification

An N domain construct D629 cloned into the vector pECE,32 encoding the first 629 residues of somatic ACE, was subcloned into pBlueScript via XbaI and EcoRI and sequenced. D629 was subcloned into pcDNA3.1(+) using the same restriction sites. This N domain construct was then subsequently introduced into the CHO cell glutamine synthetase (GS) expression vector pEE14 using HindIII and XbaI. The identity of the construct and its correct orientation was confirmed via restriction enzyme digests.

CHO-K1

Acknowledgements

We thank the staff at the Synchrotron Radiation Source, Daresbury (UK) for their support during X-ray data collection. This work was supported by the Wellcome Trust, UK (Project Grant 071047 to K.R.A., a Senior International Research Fellowship 070060 to E.D.S.) and the National Research Foundation, South Africa (grant to E.D.S.). The authors declare that they do not have conflicting financial interests.

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