Proteomic Analysis of Human Macrophages Overexpressing Angiotensin-Converting Enzyme.

Angiotensin converting enzyme (ACE) exerts strong modulation of myeloid cell function independently of its cardiovascular arm. The success of the ACE-overexpressing murine macrophage model, ACE 10/10, in treating microbial infections and cancer opens a new avenue into whether ACE overexpression in human macrophages shares these benefits. Additionally, as ACE inhibitors are a widely used antihypertensive medication, their impact on ACE expressing immune cells is of interest and currently understudied. In the present study, we utilized mass spectrometry to characterize and assess global proteomic changes in an ACE-overexpressing human THP-1 cell line. Additionally, proteomic changes and cellular uptake following treatment with an ACE C-domain selective inhibitor, lisinopril-tryptophan, were also assessed. ACE activity was significantly reduced following inhibitor treatment, despite limited uptake within the cell, and both RNA processing and immune pathways were significantly dysregulated with treatment. Also present were upregulated energy and TCA cycle proteins and dysregulated cytokine and interleukin signaling proteins with ACE overexpression. A novel, functionally enriched immune pathway that appeared both with ACE overexpression and inhibitor treatment was neutrophil degranulation. ACE overexpression within human macrophages showed similarities with ACE 10/10 murine macrophages, paving the way for mechanistic studies aimed at understanding the altered immune function.

Structural insights into the inhibitory mechanism of angiotensin-I-converting enzyme by the lactotripeptides IPP and VPP.

Human somatic angiotensin-1-converting enzyme (sACE) is composed of a catalytic N-(nACE) and C-domain (cACE) of similar size with different substrate specificities. It is involved in the regulation of blood pressure by converting angiotensin I to the vasoconstrictor angiotensin II and has been a major focus in the development of therapeutics for hypertension. Bioactive peptides from various sources, including milk, have been identified as natural ACE inhibitors. We report the structural basis for the role of two lacototripeptides, Val-Pro-Pro and Ile-Pro-Pro, in domain-specific inhibition of ACE using X-ray crystallography and kinetic analysis. The lactotripeptides have preference for nACE due to altered polar interactions distal to the catalytic zinc ion. Elucidating the mechanism of binding and domain selectivity of these peptides also provides important insights into the functional roles of ACE.

Peptide inhibitors of angiotensin-I converting enzyme based on angiotensin (1-7) with selectivity for the C-terminal domain.

The renin-angiotensin system (RAS) is a key regulator of human arterial pressure. Several of its effects are modulated by angiotensin II, an octapeptide originating from the action of angiotensin-I converting enzyme (ACE) on the decapeptide angiotensin-I. ACE possess two active sites (nACE and cACE) that have their own kinetic and substrate specificities. ACE inhibitors are widely used as the first-line treatment for hypertension and other heart-related diseases, but because they inactivate both ACE domains, their use is associated with serious side effects. Thus, the search for domain-specific ACE inhibitors has been the focus of intense research. Angiotensin (1-7), a peptide that also belongs to the RAS, acts as a substrate of nACE and an inhibitor of cACE. We have synthetized 15 derivatives of Ang (1-7), sequentially removing the N-terminal amino acids and modifying peptides extremities, to find molecules with improved selectivity and inhibition properties. Ac-Ang (2-7)-NH2 is a good ACE inhibitor, resistant to cleavage and with improved cACE selectivity. Molecular dynamics simulations provided a model for this peptide's selectivity, due to Val3 and Tyr4 interactions with ACE subsites. Val3 has an important interaction with the S3 subsite, since its removal greatly reduced peptide-enzyme interactions. Taken together, our findings support ongoing studies using insights from the binding of Ac-Ang (2-7)-NH2 to develop effective cACE inhibitors.

The Angiotensin AT2 Receptor: From a Binding Site to a Novel Therapeutic Target.

Discovered more than 30 years ago, the angiotensin AT2 receptor (AT2R) has evolved from a binding site with unknown function to a firmly established major effector within the protective arm of the renin-angiotensin system (RAS) and a target for new drugs in development. The AT2R represents an endogenous protective mechanism that can be manipulated in the majority of preclinical models to alleviate lung, renal, cardiovascular, metabolic, cutaneous, and neural diseases as well as cancer. This article is a comprehensive review summarizing our current knowledge of the AT2R, from its discovery to its position within the RAS and its overall functions. This is followed by an in-depth look at the characteristics of the AT2R, including its structure, intracellular signaling, homo- and heterodimerization, and expression. AT2R-selective ligands, from endogenous peptides to synthetic peptides and nonpeptide molecules that are used as research tools, are discussed. Finally, we summarize the known physiological roles of the AT2R and its abundant protective effects in multiple experimental disease models and expound on AT2R ligands that are undergoing development for clinical use. The present review highlights the controversial aspects and gaps in our knowledge of this receptor and illuminates future perspectives for AT2R research. SIGNIFICANCE STATEMENT: The angiotensin AT2 receptor (AT2R) is now regarded as a fully functional and important component of the renin-angiotensin system, with the potential of exerting protective actions in a variety of diseases. This review provides an in-depth view of the AT2R, which has progressed from being an enigma to becoming a therapeutic target.

Exploring the Impact of ACE Inhibition in Immunity and Disease.

Angiotensin-converting enzyme (ACE) is a zinc-dependent dipeptidyl carboxypeptidase and is crucial in the renin-angiotensin-aldosterone system (RAAS) but also implicated in immune regulation. Intrinsic ACE has been detected in several immune cell populations, including macrophages and neutrophils, where its overexpression results in enhanced bactericidal and antitumour responses, independent of angiotensin II. With roles in antigen presentation and inflammation, the impact of ACE inhibitors must be explored to understand how ACE inhibition may impact our ability to clear infections or malignancy, particularly in the wake of the coronavirus (SARS-CoV2) pandemic and as antibiotic resistance grows. Patients using ACE inhibitors may be more at risk of postsurgical complications as ACE inhibition in human neutrophils results in decreased ROS and phagocytosis whilst angiotensin receptor blockers (ARBs) have no effect. In contrast, ACE is also elevated in certain autoimmune diseases such as rheumatoid arthritis and lupus, and its inhibition benefits patient outcome where inflammatory immune cells are overactive. Although the ACE autoimmune landscape is changing, some studies have conflicting results and require further input. This review seeks to highlight the need for further research covering ACE inhibitor therapeutics and their potential role in improving autoimmune conditions, cancer, or how they may contribute to immunocompromise during infection and neurodegenerative diseases. Understanding ACE inhibition in immune cells is a developing field that will alter how ACE inhibitors are designed in future and aid in developing therapeutic interventions.

Cryo-EM reveals mechanisms of angiotensin I-converting enzyme allostery and dimerization.

Hypertension (high blood pressure) is a major risk factor for cardiovascular disease, which is the leading cause of death worldwide. The somatic isoform of angiotensin I-converting enzyme (sACE) plays a critical role in blood pressure regulation, and ACE inhibitors are thus widely used to treat hypertension and cardiovascular disease. Our current understanding of sACE structure, dynamics, function, and inhibition has been limited because truncated, minimally glycosylated forms of sACE are typically used for X-ray crystallography and molecular dynamics simulations. Here, we report the first cryo-EM structures of full-length, glycosylated, soluble sACE (sACES1211 ). Both monomeric and dimeric forms of the highly flexible apo enzyme were reconstructed from a single dataset. The N- and C-terminal domains of monomeric sACES1211 were resolved at 3.7 and 4.1 Å, respectively, while the interacting N-terminal domains responsible for dimer formation were resolved at 3.8 Å. Mechanisms are proposed for intradomain hinging, cooperativity, and homodimerization. Furthermore, the observation that both domains were in the open conformation has implications for the design of sACE modulators.

Structural basis for the inhibition of human angiotensin-1 converting enzyme by fosinoprilat.

Human angiotensin I-converting enzyme (ACE) has two isoforms, somatic ACE (sACE) and testis ACE (tACE). The functions of sACE are widespread, with its involvement in blood pressure regulation most extensively studied. sACE is composed of an N-domain (nACE) and a C-domain (cACE), both catalytically active but have significant structural differences, resulting in different substrate specificities. Even though ACE inhibitors are used clinically, they need much improvement because of serious side effects seen in patients (~ 25-30%) with long-term treatment due to nonselective inhibition of nACE and cACE. Investigation into the distinguishing structural features of each domain is therefore of vital importance for the development of domain-specific inhibitors with minimal side effects. Here, we report kinetic data and high-resolution crystal structures of both nACE (1.75 Å) and cACE (1.85 Å) in complex with fosinoprilat, a clinically used inhibitor. These structures allowed detailed analysis of the molecular features conferring domain selectivity by fosinoprilat. Particularly, altered hydrophobic interactions were observed to be a contributing factor. These experimental data contribute to improved understanding of the structural features that dictate ACE inhibitor domain selectivity, allowing further progress towards designing novel 2nd-generation domain-specific potent ACE inhibitors suitable for clinical administration, with a variety of potential future therapeutic benefits. DATABASE: The atomic coordinates and structure factors for nACE-fosinoprilat and cACE-fosinoprilat structures have been deposited with codes 7Z6Z and 7Z70, respectively, in the RCSB Protein Data Bank, www.pdb.org.

Probing the Requirements for Dual Angiotensin-Converting Enzyme C-Domain Selective/Neprilysin Inhibition.

Selective inhibition of the angiotensin-converting enzyme C-domain (cACE) and neprilysin (NEP), leaving the ACE N-domain (nACE) free to degrade bradykinin and other peptides, has the potential to provide the potent antihypertensive and cardioprotective benefits observed for nonselective dual ACE/NEP inhibitors, such as omapatrilat, without the increased risk of adverse effects. We have synthesized three 1-carboxy-3-phenylpropyl dipeptide inhibitors with nanomolar potency based on the previously reported C-domain selective ACE inhibitor lisinopril-tryptophan (LisW) to probe the structural requirements for potent dual cACE/NEP inhibition. Here we report the synthesis, enzyme kinetic data, and high-resolution crystal structures of these inhibitors bound to nACE and cACE, providing valuable insight into the factors driving potency and selectivity. Overall, these results highlight the importance of the interplay between the S1' and S2' subsites for ACE domain selectivity, providing guidance for future chemistry efforts toward the development of dual cACE/NEP inhibitors.

Targeting the oncogenic TBX3:nucleolin complex to treat multiple sarcoma subtypes.

Sarcomas are diverse cancers of mesenchymal origin, with compromised clinical management caused by insufficient diagnostic biomarkers and limited treatment options. The transcription factor TBX3 is upregulated in a diverse range of sarcoma subtypes, where it plays a direct oncogenic role, and it may thus represent a novel therapeutic target. To identify versatile ways to target TBX3, we performed affinity purification coupled by mass spectrometry to identify putative TBX3 protein cofactors that regulate its oncogenic activity in sarcomas. Here we identify and validate the multifunctional phosphoprotein nucleolin as a TBX3 cofactor. We show that nucleolin is co-expressed with TBX3 in several sarcoma subtypes and their expression levels positively correlate in sarcoma patients which are associated with poor prognosis. Furthermore, we demonstrate that nucleolin and TBX3 interact in chondrosarcoma, liposarcoma and rhabdomyosarcoma cells where they act together to enhance proliferation and migration and regulate a common set of tumor suppressor genes. Importantly, the nucleolin targeting aptamer, AS1411, exhibits selective anti-cancer activity in these cells and mislocalizes TBX3 and nucleolin to the cytoplasm which correlates with the re-expression of the TBX3/nucleolin target tumor suppressors CDKN1A (p21CIP1) and CDKN2A (p14ARF). Our findings provide the first evidence that TBX3 requires nucleolin to promote features of sarcomagenesis and that disruption of the oncogenic TBX3-nucleolin interaction by AS1411 may be a novel approach for treating sarcomas.

Investigating the antifibrotic potential of N-acetyl seryl-aspartyl-lysyl-proline sequence peptides.

N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is a physiological antifibrotic peptide that is hydrolysed by angiotensin I-converting enzyme (ACE). The beneficial antifibrotic effects of ACE inhibitors have been attributed, in part, to its inhibition of Ac-SDKP cleavage. There is indirect evidence that the SDK fragment of Ac-SDKP is the main component required for its antiproliferative action. However, the exact component of the physiological peptide that is responsible for its antifibrotic effect has yet to be determined. Ac-SDKP-derived analogues that are resistant to ACE degradation may provide a new avenue for fibrosis therapy. We tested the antifibrotic potential of various Ac-SDKP peptide sequences and an analogue resistant to ACE degradation in lung fibroblasts. We investigated the contribution and molecular mechanism of action of the amino acid residues in the Ac-SDKP sequence to its antifibrotic effects, and the effects of Ac-SDKP peptides in the prevention of collagen deposition in cells. The Ac-DKP fragment moderately inhibited endothelin-1 (ET-1) mediated transforming growth factor-ß (TGF- ß) expression, and could be slowly cleaved by ACE, revealing a different sequence requirement for the antifibrotic action of Ac-SDKP. The Ac-SDψKP analogue (where the peptide bond between the aspartate and lysine is reduced) inhibited TGF-ß/small mother against decapentaplegic (Smad)-3 signalling and collagen deposition. The Ac-SDKP peptide, in combination with ACEi, demonstrated a greater inhibition of hydroxyproline as compared to Ac-SDKP alone.

Selective Inhibition of the C-Domain of ACE (Angiotensin-Converting Enzyme) Combined With Inhibition of NEP (Neprilysin): A Potential New Therapy for Hypertension.

[Figure: see text].

Epitope mapping of novel monoclonal antibodies to human angiotensin I-converting enzyme.

Angiotensin I-converting enzyme (ACE, CD143) plays a crucial role in blood pressure regulation, vascular remodeling, and immunity. A wide spectrum of mAbs to different epitopes on the N and C domains of human ACE have been generated and used to study different aspects of ACE biology, including establishing a novel approach-conformational fingerprinting. Here we characterized a novel set of 14 mAbs, developed against human seminal fluid ACE. The epitopes for these novel mAbs were defined using recombinant ACE constructs with truncated N and C domains, species cross-reactivity, ACE mutagenesis, and competition with the previously mapped anti-ACE mAbs. Nine mAbs recognized regions on the N domain, and 5 mAbs-on the C domain of ACE. The epitopes for most of these novel mAbs partially overlap with epitopes mapped onto ACE by the previously generated mAbs, whereas mAb 8H1 recognized yet unmapped region on the C domain where three ACE mutations associated with Alzheimer's disease are localized and is a marker for ACE mutation T877M. mAb 2H4 could be considered as a specific marker for ACE in dendritic cells. This novel set of mAbs can identify even subtle changes in human ACE conformation caused by tissue-specific glycosylation of ACE or mutations, and can detect human somatic and testicular ACE in biological fluids and tissues. Furthermore, the high reactivity of these novel mAbs provides an opportunity to study changes in the pattern of ACE expression or glycosylation in different tissues, cells, and diseases, such as sarcoidosis and Alzheimer's disease.

Novel ACE mutations mimicking sarcoidosis by increasing blood ACE levels.

An elevated blood angiotensin I-converting enzyme (ACE) supports diagnosis of sarcoidosis and Gaucher disease. However, some ACE mutations increase ACE shedding, and patients with these mutations are therefore at risk of being incorrectly diagnosed with sarcoidosis because of elevated serum ACE levels. We applied a novel approach called "ACE phenotyping" to identify possible ACE mutations in 3 pulmonary clinic patients that had suspected sarcoidosis based on elevated blood ACE levels. Conformational fingerprinting of ACE indicated that these mutations may be localized in the stalk region of the protein and these were confirmed by whole exome sequencing. Index patient 1 (IP1) had a mutation (P1199L) that had been previously identified, while the other 2 patients had novel ACE mutations. IP2 had 2 mutations, T887M and N1196K (eliminating a putative glycosylation site), while IP3 had a stop codon mutation Q1124X (eliminating the transmembrane anchor). We also performed a comprehensive analysis of the existing database of all ACE mutations to estimate the proportion of mutations increasing ACE shedding. The frequency of ACE mutations resulting in increased blood ACE levels may be much higher than previously estimated. ACE phenotyping, together with whole exome sequencing, is a diagnostic approach that could prevent unnecessary invasive and/or costly diagnostic procedures, or potentially harmful treatment for patients misdiagnosed on the basis of elevated blood ACE levels.

Angiotensin-converting enzyme open for business: structural insights into the subdomain dynamics.

Angiotensin-1-converting enzyme (ACE) is a key enzyme in the renin-angiotensin-aldosterone and kinin systems where it cleaves angiotensin I and bradykinin peptides, respectively. However, ACE also participates in numerous other physiological functions, can hydrolyse many peptide substrates and has various exo- and endopeptidase activities. ACE achieves this complexity by containing two homologous catalytic domains (N- and C-domains), which exhibit different substrate specificities. Here, we present the first open conformation structures of ACE N-domain and a unique closed C-domain structure (2.0 Å) where the C terminus of a symmetry-related molecule is observed inserted into the active-site cavity and binding to the zinc ion. The open native N-domain structure (1.85 Å) enables comparison with ACE2, a homologue previously observed in open and closed states. An open S2 _S'-mutant N-domain structure (2.80 Å) includes mutated residues in the S2 and S' subsites that effect ligand binding, but are distal to the binding site. Analysis of these structures provides important insights into how structural features of the ACE domains are able to accommodate the wide variety of substrates and allow different peptidase activities. DATABASE: The atomic coordinates and structure factors for Open nACE, Open S2_S'-nACE and Native G13-cACE structures have been deposited with codes 6ZPQ, 6ZPT and 6ZPU, respectively, in the RCSB Protein Data Bank, www.pdb.org.

ACE2 and ACE: structure-based insights into mechanism, regulation and receptor recognition by SARS-CoV.

Angiotensin converting enzyme (ACE) is well-known for its role in blood pressure regulation via the renin-angiotensin aldosterone system (RAAS) but also functions in fertility, immunity, haematopoiesis and diseases such as obesity, fibrosis and Alzheimer's dementia. Like ACE, the human homologue ACE2 is also involved in blood pressure regulation and cleaves a range of substrates involved in different physiological processes. Importantly, it is the functional receptor for severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2 responsible for the 2020, coronavirus infectious disease 2019 (COVID-19) pandemic. Understanding the interaction between SARS-CoV-2 and ACE2 is crucial for the design of therapies to combat this disease. This review provides a comparative analysis of methodologies and findings to describe how structural biology techniques like X-ray crystallography and cryo-electron microscopy have enabled remarkable discoveries into the structure-function relationship of ACE and ACE2. This, in turn, has enabled the development of ACE inhibitors for the treatment of cardiovascular disease and candidate therapies for the treatment of COVID-19. However, despite these advances the function of ACE homologues in non-human organisms is not yet fully understood. ACE homologues have been discovered in the tissues, body fluids and venom of species from diverse lineages and are known to have important functions in fertility, envenoming and insect-host defence mechanisms. We, therefore, further highlight the need for structural insight into insect and venom ACE homologues for the potential development of novel anti-venoms and insecticides.

Prospects for SARS-CoV-2 diagnostics, therapeutics and vaccines in Africa.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a global pandemic, prompting unprecedented efforts to contain the virus. Many developed countries have implemented widespread testing and have rapidly mobilized research programmes to develop vaccines and therapeutics. However, these approaches may be impractical in Africa, where the infrastructure for testing is poorly developed and owing to the limited manufacturing capacity to produce pharmaceuticals. Furthermore, a large burden of HIV-1 and tuberculosis in Africa could exacerbate the severity of infection and may affect vaccine immunogenicity. This Review discusses global efforts to develop diagnostics, therapeutics and vaccines, with these considerations in mind. We also highlight vaccine and diagnostic production platforms that are being developed in Africa and that could be translated into clinical development through appropriate partnerships for manufacture.

Molecular Basis for Omapatrilat and Sampatrilat Binding to Neprilysin-Implications for Dual Inhibitor Design with Angiotensin-Converting Enzyme.

Neprilysin (NEP) and angiotensin-converting enzyme (ACE) are two key zinc-dependent metallopeptidases in the natriuretic peptide and kinin systems and renin-angiotensin-aldosterone system, respectively. They play an important role in blood pressure regulation and reducing the risk of heart failure. Vasopeptidase inhibitors omapatrilat and sampatrilat possess dual activity against these enzymes by blocking the ACE-dependent conversion of angiotensin I to the potent vasoconstrictor angiotensin II while simultaneously halting the NEP-dependent degradation of vasodilator atrial natriuretic peptide. Here, we report crystal structures of omapatrilat, sampatrilat, and sampatrilat-ASP (a sampatrilat analogue) in complex with NEP at 1.75, 2.65, and 2.6 Å, respectively. A detailed analysis of these structures and the corresponding structures of ACE with these inhibitors has provided the molecular basis of dual inhibitor recognition involving the catalytic site in both enzymes. This new information will be very useful in the design of safer and more selective vasopeptidase inhibitors of NEP and ACE for effective treatment in hypertension and heart failure.

ACE-domain selectivity extends beyond direct interacting residues at the active site.

Angiotensin-converting enzyme (ACE) is best known for its formation of the vasopressor angiotensin II that controls blood pressure but is also involved in other physiological functions through the hydrolysis of a variety of peptide substrates. The enzyme contains two catalytic domains (nACE and cACE) that have different affinities for ACE substrates and inhibitors. We investigated whether nACE inhibitor backbones contain a unique property which allows them to take advantage of the hinging of nACE. Kinetic analysis showed that mutation of unique nACE residues, in both the S2 pocket and around the prime subsites (S') to their C-domain counterparts, each resulted in a decrease in the affinity of nACE specific inhibitors (SG6, 33RE and ketoACE-13) but it required the combined S2_S' mutant to abrogate nACE-selectivity. However, this was not observed with the non-domain-selective inhibitors enalaprilat and omapatrilat. High-resolution structures were determined for the minimally glycosylated nACE with the combined S2_S' mutations in complex with the ACE inhibitors 33RE (1.8 Å), omapatrilat (1.8 Å) and SG6 (1.7 Å). These confirmed that the affinities of the nACE-selective SG6, 33RE and ketoACE-13 are not only affected by direct interactions with the immediate environment of the binding site, but also by more distal residues. This study provides evidence for a more general mechanism of ACE inhibition involving synergistic effects of not only the S2, S1' and S2' subsites, but also residues involved in the sub-domain interface that effect the unique ways in which the two domains stabilize active site loops to favour inhibitor binding.

Novel Therapeutic Approaches Targeting the Renin-Angiotensin System and Associated Peptides in Hypertension and Heart Failure.

Despite the success of renin-angiotensin system (RAS) blockade by angiotensin-converting enzyme (ACE) inhibitors and angiotensin II type 1 receptor (AT1R) blockers, current therapies for hypertension and related cardiovascular diseases are still inadequate. Identification of additional components of the RAS and associated vasoactive pathways, as well as new structural and functional insights into established targets, have led to novel therapeutic approaches with the potential to provide improved cardiovascular protection and better blood pressure control and/or reduced adverse side effects. The simultaneous modulation of several neurohumoral mediators in key interconnected blood pressure-regulating pathways has been an attractive approach to improve treatment efficacy, and several novel approaches involve combination therapy or dual-acting agents. In addition, increased understanding of the complexity of the RAS has led to novel approaches aimed at upregulating the ACE2/angiotensin-(1-7)/Mas axis to counter-regulate the harmful effects of the ACE/angiotensin II/angiotensin III/AT1R axis. These advances have opened new avenues for the development of novel drugs targeting the RAS to better treat hypertension and heart failure. Here we focus on new therapies in preclinical and early clinical stages of development, including novel small molecule inhibitors and receptor agonists/antagonists, less conventional strategies such as gene therapy to suppress angiotensinogen at the RNA level, recombinant ACE2 protein, and novel bispecific designer peptides.

Interacting cogs in the machinery of the renin angiotensin system.

Somatic angiotensin converting enzyme (sACE) is well-known for its role in blood pressure regulation and consequently, ACE inhibitors are widely prescribed for the treatment of hypertension. More than 60 years after the discovery of sACE, however, the molecular details of its substrate hydrolysis and inhibition are still poorly understood. Isothermal titration calorimetry, molecular dynamics simulations and fine epitope mapping suggest that substrate or inhibitor binding triggers a hinging motion between the two subdomains of each domain. Ligand binding to one domain further induces a conformational change in sACE to negatively affect the second domain's function and can also cause dimerization between sACE molecules. This has been linked to an increase in sACE expression via intracellular signalling. Inhibitor-induced dimerization could thus decrease the efficacy of hypertension treatment. At present, the only structural information available for sACE are crystal structures of the truncated domains in the closed conformation due to the presence of ligands. These structures do not provide any information regarding the open active site conformation prior to ligand binding, the relative orientation of the two domains in full-length sACE, or the dimerization interface. To guarantee effective therapeutic intervention, further research is required to investigate the hinging, negative cooperativity and dimerization of sACE. This review describes our current understanding of these interactions and proposes how recent advances in cryo-electron microscopy could enable structural elucidation of their mechanisms.