Crystal structure of a variable region segment of Leptospira host-interacting outer surface protein, LigA, reveals the orientation of Ig-like domains.

Leptospiral immunoglobulin-like (Lig) protein family is a surface-exposed protein from the pathogenic Leptospira. The Lig protein family has been identified as an essential virulence factor of L. interrogan. One of the family members, LigA, contains 13 homologous tandem repeats of bacterial Ig-like (Big) domains in its extracellular portion. It is crucial in binding with the host's Extracellular matrices (ECM) and complement factors. However, its vital role in the invasion and evasion of pathogenic Leptospira, structural details, and domain organization of the extracellular portion of this protein are not explored thoroughly. Here, we described the first high-resolution crystal structure of a variable region segment (LigA8-9) of LigA at 1.87 Å resolution. The structure showed some remarkably distinctive aspects compared with other closely related Immunoglobulin domains. The structure illustrated the relative orientation of two domains and highlighted the role of the linker region in the domain orientation. We also observed an apparent electron density of Ca2+ ions coordinated with a proper interacting geometry within the protein. Molecular dynamic simulations demonstrated the involvement of a linker salt bridge in providing rigidity between the two domains. Our study proposes an overall arrangement of Ig-like domains in the LigA protein. The structural understanding of the extracellular portion of LigA and its interaction with the ECM provides insight into developing new therapeutics directed toward leptospirosis.

Biochemical characterization, substrate and stereoselectivity of an outer surface putative α/ß hydrolase from the pathogenic Leptospira.

The genome of pathogenic leptospira encodes a plethora of outer surface and secretory proteins. The outer surface or secreted α/ß hydrolases in a few pathogenic organisms are crucial virulent factors. They hydrolyze host immune factors and pathogen's immune-activating ligands, which help pathogens to evade the host's innate immunity. In this study, we report biochemical characterizations, substrate and stereoselectivity of one of the leptospiral outer surface putative α/ß hydrolases, IQB77_09235 (LABH). Purified LABH displayed better kinetic parameters towards small water-soluble esters such as p-nitrophenyl acetate and p-nitrophenyl butyrate. The LABH exhibited moderate thermostability and displayed a pH optimum of 8.5. Remarkably, a phylogenetic study suggested that LABH does not cluster with other characterized bacterial esterases or lipases. Protein structural modeling revealed that some structural features are closely associated with Staphylococcus hycus lipase (SAH), a triacylglycerol hydrolase. The hydrolytic activity of the protein was found to be inhibited by a lipase inhibitor, orlistat. Biocatalytic application of the protein in the kinetic resolution of racemic 1-phenylethyl acetate reveals excellent enantioselectivity (E > 500) in the production of (R)-1-phenylethanol, a valuable chiral synthon in several industries. To our knowledge, this is the first detailed characterization of outer surface α/ß hydrolases from leptospiral spp.

Multiepitope-based vaccine design by exploring antigenic potential among leptospiral lipoproteins using comprehensive immunoinformatics and structure-based approaches.

Leptospirosis is a tropical and globally neglected zoonotic disease caused by pathogenic spirochetes, Leptospira. Although the disease has been studied for decades, a potent or effective vaccine is not available so far. Efforts are being made to design an efficient vaccine candidate using different approaches. Immunoinformatics approaches have been proven to be promising in terms of time and cost. Here, we used immunoinformatics and structure-based approaches to evaluate antigenic B- and T-cell epitopes present on the leptospiral lipoproteins (LipL). The promiscuous overlapping epitopes (B-cell, T-cell, interferon (IFN)-γ positive, and non-allergens), which can induce humoral, cell-mediated, and innate immunity, were selected to generate a multiepitope chimeric vaccine. To enhance the vaccine immunogenicity, a Toll-like receptor (TLR) agonist was fused to the vaccine with a suitable linker. The chimeric vaccine structure was predicted for molecular docking studies with immune receptors. Moreover, the stability of the vaccine-immune receptor complexes was analyzed by normal mode analysis (NMA). The potency of the vaccine construct was predicted by the immune simulation tool. The study provides additional information toward constructing peptide-based chimeric vaccines  against Leptospira.

Characterization of novel nuclease and protease activities among Leptospiral immunoglobulin-like proteins.

Bacterial immunoglobulin-like (BIg) domain containing proteins play a variety of biological functions. Leptospiral Immunoglobulin-like (Lig) proteins are well-known virulence factors located on the surface of the pathogenic Leptospira that act during adhesion, invasion, and immune evasion. The Lig proteins have many roles and have been designated as multifaceted proteins. However, the hydrolyzing function of Lig proteins is not yet investigated in detail. Here, we report novel in-vitro nuclease and protease activities in the Ig-like domain of LigA protein. All Ig-like domains were able to cleave DNA in the presence of a divalent ion, but not RNA. Site-directed mutagenesis revealed Mg+2 binding residues in the Ig-like domain of LigA7. The basis of novel nuclease activity may be associated with protein adopting different conformation in the presence of divalent ions and substrate as investigated by change of intrinsic fluorescence. The docking of a stretch of double-strand DNA shows the binding on the positive surface of the protein. In addition, the protein is also observed to cleave a general protease substrate, ß-casein, in our experimental condition. Our results proposed that the novel functions may be associated with neutrophil extracellular Trap (NET) evasion. Overall this study enhances the basic knowledge of non-nuclease proteins involved in the DNA cleavage activity and makes the foundation to explore its in-vivo activity in pathogenic Leptospira and other pathogens as well. Moreover, this information may be utilized to develop preventive strategies to interfere with Leptospira immune evasion.

Probing structural basis for enhanced binding of SARS-CoV-2 P.1 variant spike protein with the human ACE2 receptor.

The initial step of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) involves the binding of receptor binding domain (RBD) of the spike protein to the angiotensin converting enzyme 2 (ACE2) receptor. Each successive wave of SARS-CoV-2 reports emergence of many new variants, which is associated with mutations in the RBD as well as other parts of the spike protein. These mutations are reported to have enhanced affinity towards the ACE2 receptor as well as are also crucial for the virus transmission. Many computational and experimental studies have demonstrated the effect of individual mutation on the RBD-ACE2 binding. However, the cumulative effect of mutations on the RBD and away from the RBD was not investigated in detail. We report here a comparative analysis on the structural communication and dynamics of the RBD and truncated S1 domain of spike protein in complex with the ACE2 receptor from SARS-CoV-2 wild type and its P.1 variant. Our integrative network and dynamics approaches highlighted a subtle conformational changes in the RBD as well as truncated S1 domain of spike protein at the protein contact level, responsible for the increased affinity with the ACE2 receptor. Moreover, our study also identified the commonalities and differences in the dynamics of the interactions between spike protein of SARS-CoV-2 wild type and its P.1 variant with the ACE2 receptor. Further, our investigation yielded an understanding towards identification of the unique RBD residues crucial for the interaction with the ACE2 host receptor. Overall, the study provides an insight for designing better therapeutics against the circulating P.1 variants as well as other future variants.

Enzyme engineering improves catalytic efficiency and enantioselectivity of hydroxynitrile lyase for promiscuous retro-nitroaldolase activity.

Protein engineering to improve promiscuous catalytic activity is important for biocatalytic application of enzymes in green synthesis. We uncovered the significance of binding site residues in Arabidopsis thaliana hydroxynitrile lyase (AtHNL) for promiscuous retro-nitroaldolase activity. Engineering of AtHNL has improved enantioselective retro-nitroaldolase activity, a synthetically important biotransformation, for the production of enantiopure ß-nitroalcohols having absolute configuration opposite to that of the stereopreference of the HNL. The variant F179A has shown âˆ¼ 12 fold increased selectivity towards the retro-nitroaldol reaction over cyanogenesis, the natural activity of the parent enzyme. Screening of the two saturation libraries of Phe179 and Tyr14 revealed several variants with higher kcat, while F179N showed âˆ¼ 2.4-fold kcat/Km than the native enzyme towards retro-nitroaldol reaction. Variants F179N, F179M, F179W, F179V, F179I, Y14L, and Y14M have shown > 99% ee in the preparation of (S)-2-nitro-1-phenylethanol (NPE) from the racemic substrate, while F179N has shown the E value of 138 vs. 81 by the wild type. Our molecular docking and dynamics simulations (MDS) studies results provided insights into the molecular basis of higher enantioselectivity by the F179N toward the retro-nitroaldolase activity than the other mutants. Binding energy calculations also showed the higher negative binding free energy in the case of F179N-(R)-NPE compared to other complexes that support our experimental low Km by the F179N for NPE. A plausible retro-nitroaldol reaction mechanism was proposed based on the MDS study of enzyme-substrate interaction.

Immunoinformatics-Based Designing of a Multi-Epitope Chimeric Vaccine From Multi-Domain Outer Surface Antigens of Leptospira.

Accurate information on antigenic epitopes within a multi-domain antigen would provide insights into vaccine design and immunotherapy. The multi-domain outer surface Leptospira immunoglobulin-like (Lig) proteins LigA and LigB, consisting of 12-13 homologous bacterial Ig (Big)-like domains, are potential antigens of Leptospira interrogans. Currently, no effective vaccine is available against pathogenic Leptospira. Both the humoral immunity and cell-mediated immunity of the host play critical roles in defending against Leptospira infection. Here, we used immunoinformatics approaches to evaluate antigenic B-cell lymphocyte (BCL) and cytotoxic T-lymphocyte (CTL) epitopes from Lig proteins. Based on certain crucial parameters, potential epitopes that can stimulate both types of adaptive immune responses were selected to design a chimeric vaccine construct. Additionally, an adjuvant, the mycobacterial heparin-binding hemagglutinin adhesin (HBHA), was incorporated into the final multi-epitope vaccine construct with a suitable linker. The final construct was further scored for its antigenicity, allergenicity, and physicochemical parameters. A three-dimensional (3D) modeled construct of the vaccine was implied to interact with Toll-like receptor 4 (TLR4) using molecular docking. The stability of the vaccine construct with TLR4 was predicted with molecular dynamics simulation. Our results demonstrate the application of immunoinformatics and structure biology strategies to develop an epitope-specific chimeric vaccine from multi-domain proteins. The current findings will be useful for future experimental validation to ratify the immunogenicity of the chimera.

Phosphorylation of eukaryotic initiation factor eIFiso4E enhances the binding rates to VPg of turnip mosaic virus.

Binding of phosphorylated eIFiso4E with viral genome-linked protein (VPg) of turnip mosaic virus was examined by stopped-flow, fluorescence, circular dichroism (CD) spectroscopy, and molecular docking analysis. Phosphorylation of eIFiso4E increased (4-fold) the binding rates as compared to unphosphorylated eIFiso4E with VPg. Stopped-flow kinetic studies of phosphorylated eIFiso4E with VPg showed a concentration-independent conformational change. The dissociation rate was about 3-fold slower for eIFiso4E∙VPg complex upon phosphorylation. Phosphorylation enhanced the association rates and lowered the dissociation rates for the eIFiso4E∙VPg binding, with having higher preferential binding to eIFiso4Ep. Binding rates for the interaction of eIFiso4Ep with VPg increased (6-fold) with an increase in temperature, 278 K to 298 K. The activation energies for binding of eIFiso4Ep and eIFiso4E with VPg were 37.2 ± 2.8 and 52.6 ± 3.6 kJ/mol, respectively. Phosphorylation decreased the activation energy for the binding of eIFiso4E to VPg. The reduced energy barrier suggests more stable platform for eIFiso4Ep∙VPg initiation complex formation, which was further supported by molecular docking analysis. Moreover, far-UV CD studies revealed that VPg formed complex with eIFiso4Ep with substantial change in the secondary structure. These results suggested that phosphorylation, not only reduced the energy barrier and dissociation rate but also enhanced binding rate, and an overall conformational change, which provides a more stable platform for efficient viral translation.

Immunoinformatics analysis of antigenic epitopes and designing of a multi-epitope peptide vaccine from putative nitro-reductases of Mycobacterium tuberculosis DosR.

Mycobacterium tuberculosis (Mtb) resides in alveolar macrophages as a non-dividing and dormant state causing latent tuberculosis. Currently, no vaccine is available against the latent tuberculosis. Latent Mtb expresses ~48 genes under the control of DosR regulon. Among these, putative nitroreductases have significantly high expression levels, help Mtb to cope up with nitrogen stresses and possess antigenic properties. In the current study, immunoinformatics methodologies are applied to predict promiscuous antigenic T-cell epitopes from putative nitro-reductases of the DosR regulon. The promiscuous antigenic T-cell epitopes prediction was performed on the basis of their potential to induce an immune response and forming a stable interaction with the HLA alleles. The highest antigenic promiscuous epitopes were assembled for designing an in-silico vaccine construct. A TLR-2 agonist Phenol-soluble modulin alpha 4 was exploited as an adjuvant. Molecular docking and Molecular Dynamics Simulations were used to predict the stability of vaccine construct with the immune receptor. The predicted promiscuous epitopes may be helpful in the construction of a subunit vaccine against latent tuberculosis, which can also be administered along with the BCG to increase its efficacy. Experimental validation is a prerequisite for the in-silico designed vaccine construct against TB infection.

Comparative protein structure network analysis on 3CLpro from SARS-CoV-1 and SARS-CoV-2.

The main protease Mpro , 3CLpro is an important target from coronaviruses. In spite of having 96% sequence identity among Mpros from SARS-CoV-1 and SARS-CoV-2; the inhibitors used to block the activity of SARS-CoV-1 Mpro so far, were found to have differential inhibitory effect on Mpro of SARS-CoV-2. The possible reason could be due to the difference of few amino acids among the peptidases. Since, overall 3-D crystallographic structure of Mpro from SARS-CoV-1 and SARS-CoV-2 is quite similar and mapping a subtle structural variation is seemingly impossible. Hence, we have attempted to study a structural comparison of SARS-CoV-1 and SARS-CoV-2 Mpro in apo and inhibitor bound states using protein structure network (PSN) based approach at contacts level. The comparative PSNs analysis of apo Mpros from SARS-CoV-1 and SARS-CoV-2 uncovers small but significant local changes occurring near the active site region and distributed throughout the structure. Additionally, we have shown how inhibitor binding perturbs the PSG and the communication pathways in Mpros . Moreover, we have also investigated the network connectivity on the quaternary structure of Mpro and identified critical residue pairs for complex formation using three centrality measurement parameters along with the modularity analysis. Taken together, these results on the comparative PSN provide an insight into conformational changes that may be used as an additional guidance towards specific drug development.

Structure-based identification of natural compound inhibitor against M. tuberculosis thioredoxin reductase: insight from molecular docking and dynamics simulation.

Antioxidant systems of M. tuberculosis (Mtb) play an important role in providing resistance in the hostile environment of mononuclear phagocytes. Thioredoxin system is a known antioxidant system that consists of three copies of thioredoxins (Trxs) and a single copy of thioredoxin reductase (TrxR). TrxR has been validated as an essential gene known to be involved in the reduction of peroxides, dinitrobenzenes and hydroperoxides, and is crucial in maintaining the survival of Mtb in macrophages. Recently, it has been demonstrated to be a druggable target. In this study, molecular docking was applied to screen more than 20,000 natural compounds from the Traditional Chinese Medicine database. Theoretical calculation of ΔGbinding by the Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) methods indicated two top-hit compounds that bind with a high affinity to the allosteric site, consisting of a hinge region, of TrxR. Further, stability and binding analysis of both compounds were carried out with molecular dynamics simulation. An analysis of conformational variation by principal component analysis (PCA) and protein contact network (PCN) uncovered the conformational changes in the compound-bound forms of protein. The NADPH domain formed many new interactions with the FAD domain in the compound-bound form, signifying that the binding may render an effect on the protein structure and function. Our results suggest that these two compounds could potentially be used for structure-based lead inhibitors against TrxR. The inhibitor selected as lead compound will be used further as a scaffold to optimize as novel anti-tuberculosis therapeutic.Communicated by Ramaswamy H. Sarma.

Comparative screening of recombinant antigen thermostability for improved leptospirosis vaccine design.

Recombinant antigens exhibit targeted protectiveproperties and offer important opportunities in the development of therapeutic technologies. Biophysical and structural methods have become important tools for the rational design and engineering of improved antigen-based vaccines. Vaccines containing Leptospira immunoglobulin-like (Lig) protein-derived antigens are currently the most promising candidates for protective immunity against the globally prevalent bacterial pathogen, Leptospira interrogans; however, vaccine trials using these domains have produced inconsistent results. Here, we compare the thermostability of domains from the main immunogenic regions from major leptospiral antigens, LigA and LigB. By measuring temperature-dependent fluorescence decay of the hydrophobic core tryptophan, 17 individual Lig protein immunoglobulin-like (Ig-like) domains were shown to display a broad range of unfolding temperatures. For a majority of the domains, stability issues begin to occur at physiologically relevant temperatures. A set of chimeric Ig-like domains was used to establish the ability of transplanted domain regions to enhance thermostability. Further insights into the determinants for domain stabilization were explored with nuclear magnetic resonance dynamics and mutational analysis. The current study has yielded a set of thermostable Ig-like domain scaffolds for use in engineering antigen-based vaccines and demonstrates the importance of incorporating thermostability screening as a design parameter.

Leptospira surface adhesin (Lsa21) induces Toll like receptor 2 and 4 mediated inflammatory responses in macrophages.

Leptospirosis is zoonotic and emerging infectious disease of global importance. Little is understood about Leptospira pathogenesis and host immune response. In the present work we have investigated how Leptospira modulates the host innate immune response mediated by Toll-like receptors (TLRs) via surface exposed proteins. We screened Leptospira outer membrane/surface proteins for their ability to activate/inhibit TLR2/4 signaling in HEK293 cell lines. Of these the 21 kDa Leptospira surface adhesin, Lsa21 had strong TLR2 and TLR4 activity leading to production of proinflammatory cytokines and expression of costimulatory molecules in mouse macrophages. This activity of Lsa21 on innate response was dependent on activation of mitogen activated protein kinases (MAPKs) via stimulating the rapid phosphorylation of p38, JNK and activation of transcription factor NF-κB. Additionally, neutralizing antibodies against TLR2 and TLR4 significantly inhibited cytokine secretion and attenuated Lsa21 induced phosphorylation of p38 and JNK. Furthermore, Lsa21 induced cytokine levels were significantly lower in TLR2-/- and TLR4-/- than in wild type mouse macrophage cell lines. Confocal microscopy and molecular docking confirmed that Lsa21 interacted with both TLR2 and TLR4. These results indicate that Lsa21 is a potent TLR2 and TLR4 agonist that induces strong innate response and may play important role in Leptospira pathogenesis.

Crystal structures of highly specific phosphinic tripeptide enantiomers in complex with the angiotensin-I converting enzyme.

Human somatic angiotensin-I converting enzyme (ACE) is a zinc-dependent dipeptidyl carboxypeptidase and a central component of the renin angiotensin aldosterone system (RAAS). Its involvement in the modulation of physiological actions of peptide hormones has positioned ACE as an important therapeutic target for the treatment of hypertension and cardiovascular disorders. Here, we report the crystal structures of the two catalytic domains of human ACE (N- and C-) in complex with FI, the S enantiomer of the phosphinic ACE/ECE-1 (endothelin converting enzyme) dual inhibitor FII, to a resolution of 1.91 and 1.85 Å, respectively. In addition, we have determined the structure of AnCE (an ACE homologue from Drosophila melanogaster) in complex with both isomers. The inhibitor FI (S configuration) can adapt to the active site of ACE catalytic domains and shows key differences in its binding mechanism mostly through the reorientation of the isoxazole phenyl side group at the P1' position compared with FII (R configuration). Differences in binding are also observed between FI and FII in complex with AnCE. Thus, the new structures of the ACE-inhibitor complexes presented here provide useful information for further exploration of ACE inhibitor pharmacophores involving phosphinic peptides and illustrate the role of chirality in enhancing drug specificity.

Molecular and thermodynamic mechanisms of the chloride-dependent human angiotensin-I-converting enzyme (ACE).

Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N- and C-domains, which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry. The x-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of Arg(522). Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu(403)-Lys(118) salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond as well as interactions of larger substrates in the S2 subsite moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate-selective nature of chloride dependence in ACE and how this varies between the N- and C-domains.

Structural basis of peptide recognition by the angiotensin-1 converting enzyme homologue AnCE from Drosophila melanogaster.

Human somatic angiotensin-1 converting enzyme (ACE) is a zinc-dependent exopeptidase, that catalyses the conversion of the decapeptide angiotensin I to the octapeptide angiotensin II, by removing a C-terminal dipeptide. It is the principal component of the renin-angiotensin-aldosterone system that regulates blood pressure. Hence it is an important therapeutic target for the treatment of hypertension and cardiovascular disorders. Here, we report the structures of an ACE homologue from Drosophila melanogaster (AnCE; a proven structural model for the more complex human ACE) co-crystallized with mammalian peptide substrates (bradykinin, Thr(6) -bradykinin, angiotensin I and a snake venom peptide inhibitor, bradykinin-potentiating peptide-b). The structures determined at 2-Å resolution illustrate that both angiotensin II (the cleaved product of angiotensin I by AnCE) and bradykinin-potentiating peptide-b bind in an analogous fashion at the active site of AnCE, but also exhibit significant differences. In addition, the binding of Arg-Pro-Pro, the cleavage product of bradykinin and Thr(6) - bradykinin, provides additional detail of the general peptide binding in AnCE. Thus the new structures of AnCE complexes presented here improves our understanding of the binding of peptides and the mechanism by which peptides inhibit this family of enzymes. DATABASE: The atomic coordinates and structure factors for AnCE-Ang II (code 4AA1), AnCE-BPPb (code 4AA2), AnCE-BK (code 4ASQ) and AnCE-Thr6-BK (code 4ASR) complexes have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/) STRUCTURED DIGITAL ABSTRACT: • AnCE cleaves Ang I by enzymatic study (View interaction) • Bradykinin and AnCE bind by x-ray crystallography (View interaction) • BPP and AnCE bind by x-ray crystallography (View interaction) • AnCE cleaves Bradykinin by enzymatic study (View interaction) • Ang II and AnCE bind by x-ray crystallography (View interaction).

Structural characterization of angiotensin I-converting enzyme in complex with a selenium analogue of captopril.

Human somatic angiotensin I-converting enzyme (ACE), a zinc-dependent dipeptidyl carboxypeptidase, is central to the regulation of the renin-angiotensin aldosterone system. It is a well-known target for combating hypertension and related cardiovascular diseases. In a recent study by Bhuyan and Mugesh [Org. Biomol. Chem. (2011) 9, 1356-1365], it was shown that the selenium analogues of captopril (a well-known clinical inhibitor of ACE) not only inhibit ACE, but also protect against peroxynitrite-mediated nitration of peptides and proteins. Here, we report the crystal structures of human testis ACE (tACE) and a homologue of ACE, known as AnCE, from Drosophila melanogaster in complex with the most promising selenium analogue of captopril (SeCap) determined at 2.4 and 2.35 Å resolution, respectively. The inhibitor binds at the active site of tACE and AnCE in an analogous fashion to that observed for captopril and provide the first examples of a protein-selenolate interaction. These new structures of tACE-SeCap and AnCE-SeCap inhibitor complexes presented here provide important information for further exploration of zinc coordinating selenium-based ACE inhibitor pharmacophores with significant antioxidant activity.

Novel mechanism of inhibition of human angiotensin-I-converting enzyme (ACE) by a highly specific phosphinic tripeptide.

Human ACE (angiotensin-I-converting enzyme) has long been regarded as an excellent target for the treatment of hypertension and related cardiovascular diseases. Highly potent inhibitors have been developed and are extensively used in the clinic. To develop inhibitors with higher therapeutic efficacy and reduced side effects, recent efforts have been directed towards the discovery of compounds able to simultaneously block more than one zinc metallopeptidase (apart from ACE) involved in blood pressure regulation in humans, such as neprilysin and ECE-1 (endothelin-converting enzyme-1). In the present paper, we show the first structures of testis ACE [C-ACE, which is identical with the C-domain of somatic ACE and the dominant domain responsible for blood pressure regulation, at 1.97Å (1 Å=0.1 nm)] and the N-domain of somatic ACE (N-ACE, at 2.15Å) in complex with a highly potent and selective dual ACE/ECE-1 inhibitor. The structural determinants revealed unique features of the binding of two molecules of the dual inhibitor in the active site of C-ACE. In both structures, the first molecule is positioned in the obligatory binding site and has a bulky bicyclic P(1)' residue with the unusual R configuration which, surprisingly, is accommodated by the large S(2)' pocket. In the C-ACE complex, the isoxazole phenyl group of the second molecule makes strong pi-pi stacking interactions with the amino benzoyl group of the first molecule locking them in a 'hand-shake' conformation. These features, for the first time, highlight the unusual architecture and flexibility of the active site of C-ACE, which could be further utilized for structure-based design of new C-ACE or vasopeptidase inhibitors.

Crystal structure of a phosphonotripeptide K-26 in complex with angiotensin converting enzyme homologue (AnCE) from Drosophila melanogaster.

Angiotensin-I converting enzyme (ACE, a zinc dependent dipeptidyl carboxypeptidase) is a major target of drugs due to its role in the modulation of blood pressure and cardiovascular disorders. Here we present a crystal structure of AnCE (an ACE homologue from Drosophila melanogaster with a single enzymatic domain) in complex with a natural product-phosphonotripeptide, K-26 at 1.96A resolution. The inhibitor binds exclusively in the S(1) and S(2) binding pockets of AnCE (coordinating the zinc ion) through ionic and hydrogen bond interactions. A detailed structural comparison of AnCE.K-26 complex with individual domains of human somatic ACE provides useful information for further exploration of ACE inhibitor pharmacophores involving phosphonic acids.

High-resolution crystal structures of Drosophila melanogaster angiotensin-converting enzyme in complex with novel inhibitors and antihypertensive drugs.

Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE.