RESUMO
Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that optimizes the peptide cargo of major histocompatibility class I (MHC-I) molecules and regulates adaptive immunity. It has unusual substrate selectivity for length and sequence, resulting in poorly understood effects on the cellular immunopeptidome. To understand substrate selection by ERAP1, we solved 2 crystal structures of the enzyme with bound transition-state pseudopeptide analogs at 1.68 Å and 1.72 Å. Both peptides have their N terminus bound at the active site and extend away along a large internal cavity, interacting with shallow pockets that can influence selectivity. The longer peptide is disordered through the central region of the cavity and has its C terminus bound in an allosteric pocket of domain IV that features a carboxypeptidase-like structural motif. These structures, along with enzymatic and computational analyses, explain how ERAP1 can select peptides based on length while retaining the broad sequence-specificity necessary for its biological function.
RESUMO
The ß3 subunit of nicotinic acetylcholine receptors (nAChRs) participates in heteropentameric assemblies with some α and other ß neuronal subunits forming a plethora of various subtypes, differing in their electrophysiological and pharmacological properties. While ß3 has for several years been considered an accessory subunit without direct participation in the formation of functional binding sites, recent electrophysiology data have disputed this notion and indicated the presence of a functional (+) side on the extracellular domain (ECD) of ß3. In this study, we present the 2.4 Å resolution crystal structure of the monomeric ß3 ECD, which revealed rather distinctive loop C features as compared to those of α nAChR subunits, leading to intramolecular stereochemical hindrance of the binding site cavity. Vigorous molecular dynamics simulations in the context of full length pentameric ß3-containing nAChRs, while not excluding the possibility of a ß3 (+) binding site, demonstrate that this site cannot efficiently accommodate the agonist nicotine. From the structural perspective, our results endorse the accessory rather than functional role of the ß3 nAChR subunit, in accordance with earlier functional studies on ß3-containing nAChRs.
Assuntos
Receptores Nicotínicos , Sítios de Ligação , Nicotina/farmacologia , Receptores Nicotínicos/metabolismoRESUMO
Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a key enzyme involved in the trimming of antigenic peptides presented by Major Histocompatibility Complex class I. It is a target of growing interest for the treatment of autoimmune diseases and in cancer immunotherapy. However, the discovery of potent and selective ERAP2 inhibitors is highly challenging. Herein, we have used kinetic target-guided synthesis (KTGS) to identify such inhibitors. Co-crystallization experiments revealed the binding mode of three different inhibitors with increasing potency and selectivity over related enzymes. Selected analogues engage ERAP2 in cells and inhibit antigen presentation in a cellular context. 4 d (BDM88951) displays favorable in vitro ADME properties and in vivo exposure. In summary, KTGS allowed the discovery of the first nanomolar and selective highly promising ERAP2 inhibitors that pave the way of the exploration of the biological roles of this enzyme and provide lead compounds for drug discovery efforts.
Assuntos
Aminopeptidases , Apresentação de Antígeno , Aminopeptidases/metabolismo , Antígenos de Histocompatibilidade Classe I , Peptídeos/metabolismoRESUMO
The pharmacological profile of medicinally relevant Ru(III) coordination compounds has been ascribed to their interactions with proteins, as several studies have provided evidence that DNA is not the primary target. In this regard, numerous spectroscopic and crystallographic studies have indicated that the Ru(III) ligands play an important role in determining the metal binding site, acting as the recognition element in the early stages of the protein-complex formation. Herein, we present a series of near-atomic-resolution X-ray crystal structures of the adducts formed between the antimetastatic metallodrug imidazolium trans-[tetrachlorido(S-dimethyl sufoxide)(1H-imidazole)ruthenate(III)] (NAMI-A) and hen egg-white lysozyme (HEWL). These structures elucidate a series of binding events starting from the noncovalent interaction of intact NAMI-A ions with HEWL (1.5 h), followed by the stepwise exchange of all Ru ligands except for 1H-imidazole (26 h) to the final "ruthenated" protein comprising one aquated Ru ion coordinated to histidine-15 of HEWL (98 h). Our structural data clearly support a two-step mechanism of protein ruthenation, illustrating the ligand-mediated recognition step of the process.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Estruturas Metalorgânicas/química , Estruturas Metalorgânicas/farmacologia , Muramidase/química , Rutênio/química , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Imidazóis/química , Modelos Moleculares , Metástase Neoplásica , Conformação ProteicaRESUMO
The high-resolution X-ray crystal structures of the adducts formed between the "half sandwich"-type Ru(II) coordination compound [RuII(1,4,7-trithiacyclononane)(ethane-1,2-diamine)Cl]+ and two proteins, namely hen egg-white lysozyme and proteinase K, are presented. The structures unveil that upon reaction with both enzymes the Ru(II) compound is coordinated by solvent-exposed aspartate residues after releasing the chloride ligand (Asp101 in lysozyme, Asp200 and Asp260 in proteinase K), while retaining the two chelating ligands. The adduct with Asp101 residue at the catalytic cleft of lysozyme is accompanied by residue-specific conformational changes to accommodate the Ru(II) fragment, whereas the complexes bound at the two calcium-binding sites of proteinase K revealed minimal structural perturbation of the enzyme. To the best of our knowledge, proteinase K is used here for the first time as a model system of protein metalation and these are the first X-ray crystal structures of protein adducts of a Ru(II) coordination compound that maintains its coordination sphere almost intact upon binding. Our data demonstrate the role of ligands in stabilizing the protein adducts via hydrophobic/aromatic or hydrogen-bonding interactions, as well as their underlying role in the selection of specific sites on the electrostatic potential surface of the enzymes.
Assuntos
Complexos de Coordenação/química , Endopeptidase K/química , Muramidase/química , Rutênio/química , Complexos de Coordenação/metabolismo , Cristalografia por Raios X , Endopeptidase K/metabolismo , Modelos Moleculares , Conformação Molecular , Muramidase/metabolismo , Rutênio/metabolismoRESUMO
In this study we report the X-ray crystal structure of the extracellular domain (ECD) of the human neuronal α2 nicotinic acetylcholine receptor (nAChR) subunit in complex with the agonist epibatidine at 3.2 Å. Interestingly, α2 was crystallized as a pentamer, revealing the intersubunit interactions in a wild type neuronal nAChR ECD and the full ligand binding pocket conferred by two adjacent α subunits. The pentameric assembly presents the conserved structural scaffold observed in homologous proteins, as well as distinctive features, providing unique structural information of the binding site between principal and complementary faces. Structure-guided mutagenesis and electrophysiological data confirmed the presence of the α2(+)/α2(-) binding site on the heteromeric low sensitivity α2ß2 nAChR and validated the functional importance of specific residues in α2 and ß2 nAChR subunits. Given the pathological importance of the α2 nAChR subunit and the high sequence identity with α4 (78%) and other neuronal nAChR subunits, our findings offer valuable information for modeling several nAChRs and ultimately for structure-based design of subtype specific drugs against the nAChR associated diseases.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/química , Subunidades Proteicas/química , Piridinas/química , Receptores Nicotínicos/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Feminino , Expressão Gênica , Humanos , Modelos Moleculares , Mutação , Oócitos/citologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Pichia/genética , Pichia/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Piridinas/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
The cell wall peptidoglycan is recognized as a primary target of the innate immune system, and usually its disintegration results in bacterial lysis. Bacillus cereus, a close relative of the highly virulent Bacillus anthracis, contains 10 polysaccharide deacetylases. Among these, the peptidoglycan N-acetylglucosamine deacetylase Bc1974 is the highest homologue to the Bacillus anthracis Ba1977 that is required for full virulence and is involved in resistance to the host's lysozyme. These metalloenzymes belong to the carbohydrate esterase family 4 (CE4) and are attractive targets for the development of new anti-infective agents. Herein we report the first X-ray crystal structures of the NodB domain of Bc1974, the conserved catalytic core of CE4s, in the unliganded form and in complex with four known metalloenzyme inhibitors and two amino acid hydroxamates that target the active site metal. These structures revealed the presence of two conformational states of a catalytic loop known as motif-4 (MT4), which were not observed previously for peptidoglycan deacetylases, but were recently shown in the structure of a Vibrio clolerae chitin deacetylase. By employing molecular docking of a substrate model, we describe a catalytic mechanism that probably involves initial binding of the substrate in a receptive, more open state of MT4 and optimal catalytic activity in the closed state of MT4, consistent with the previous observations. The ligand-bound structures presented here, in addition to the five Bc1974 inhibitors identified, provide a valuable basis for the design of antibacterial agents that target the peptidoglycan deacetylase Ba1977.
Assuntos
Amidoidrolases/química , Bacillus cereus/enzimologia , Proteínas de Bactérias/química , Amidoidrolases/antagonistas & inibidores , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Catálise , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Ligação de Hidrogênio , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Zinco/químicaRESUMO
The enantioselectivity of ß-cyclodextrin (ß-CD) towards L- and D-N-acetyltryptophan (NAcTrp) has been studied in aqueous solution and the crystalline state. NMR studies in solution show that ß-CD forms complexes of very similar but not identical geometry with both L- and D-NAcTrp and exhibits stronger binding with L-NAcTrp. In the crystalline state, only ß-CD-L-NAcTrp crystallizes readily from aqueous solutions as a dimeric complex (two hosts enclosing two guest molecules). In contrast, crystals of the complex ß-CD-D-NAcTrp were never obtained, although numerous conditions were tried. In aqueous solution, the orientation of the guest in both complexes is different than in the ß-CD-L-NAcTrp complex in the crystal. Overall, the study shows that subtle differences observed between the ß-CD-L,D-NAcTrp complexes in aqueous solution are magnified at the onset of crystallization, as a consequence of accumulation of many soft host-guest interactions and of the imposed crystallographic order, thus resulting in very dissimilar propensity of each enantiomer to produce crystals with ß-CD.
RESUMO
Endoplasmic reticulum (ER) aminopeptidases process antigenic peptide precursors to generate epitopes for presentation by MHC class I molecules and help shape the antigenic peptide repertoire and cytotoxic T-cell responses. To perform this function, ER aminopeptidases have to recognize and process a vast variety of peptide sequences. To understand how these enzymes recognize substrates, we determined crystal structures of ER aminopeptidase 2 (ERAP2) in complex with a substrate analogue and a peptidic product to 2.5 and 2.7 Å, respectively, and compared them to the apo-form structure determined to 3.0 Å. The peptides were found within the internal cavity of the enzyme with no direct access to the outside solvent. The substrate analogue extends away from the catalytic center toward the distal end of the internal cavity, making interactions with several shallow pockets along the path. A similar configuration was evident for the peptidic product, although decreasing electron density toward its C terminus indicated progressive disorder. Enzymatic analysis confirmed that visualized interactions can either positively or negatively impact in vitro trimming rates. Opportunistic side-chain interactions and lack of deep specificity pockets support a limited-selectivity model for antigenic peptide processing by ERAP2. In contrast to proposed models for the homologous ERAP1, no specific recognition of the peptide C terminus by ERAP2 was evident, consistent with functional differences in length selection and self-activation between these two enzymes. Our results suggest that ERAP2 selects substrates by sequestering them in its internal cavity and allowing opportunistic interactions to determine trimming rates, thus combining substrate permissiveness with sequence bias.
Assuntos
Aminopeptidases/metabolismo , Antígenos/metabolismo , Retículo Endoplasmático/enzimologia , Peptídeos/metabolismo , Aminopeptidases/química , Animais , Linhagem Celular , Cristalografia , Modelos Moleculares , Conformação ProteicaRESUMO
Membrane-anchored lipoproteins have a broad range of functions and play key roles in several cellular processes in Gram-positive bacteria. BA0330 and BA0331 are the only lipoproteins among the 11 known or putative polysaccharide deacetylases of Bacillus anthracis. We found that both lipoproteins exhibit unique characteristics. BA0330 and BA0331 interact with peptidoglycan, and BA0330 is important for the adaptation of the bacterium to grow in the presence of a high concentration of salt, whereas BA0331 contributes to the maintenance of a uniform cell shape. They appear not to alter the peptidoglycan structure and do not contribute to lysozyme resistance. The high resolution x-ray structure of BA0330 revealed a C-terminal domain with the typical fold of a carbohydrate esterase 4 and an N-terminal domain unique for this family, composed of a two-layered (4 + 3) ß-sandwich with structural similarity to fibronectin type 3 domains. Our data suggest that BA0330 and BA0331 have a structural role in stabilizing the cell wall of B. anthracis.
Assuntos
Amidoidrolases/metabolismo , Antraz/microbiologia , Bacillus anthracis/citologia , Bacillus anthracis/enzimologia , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Osmose/fisiologia , Estresse Fisiológico , Amidoidrolases/química , Amidoidrolases/genética , Sequência de Aminoácidos , Antraz/genética , Antraz/metabolismo , Bacillus anthracis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Western Blotting , Clonagem Molecular , Cristalografia por Raios X , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Dados de Sequência Molecular , Peptidoglicano/metabolismo , Conformação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tolerância ao Sal , Homologia de Sequência de AminoácidosRESUMO
The α9α10 nicotinic acetylcholine receptor (nAChR) was first identified in the auditory system, where it mediates synaptic transmission between efferent olivocochlear cholinergic fibers and cochlea hair cells. This receptor gained further attention due to its potential role in chronic pain and breast and lung cancers. We previously showed that α-conotoxin (α-CTx) RgIA, one of the few α9α10 selective ligands identified to date, is 300-fold less potent on human versus rat α9α10 nAChR. This species difference was conferred by only one residue in the (-), rather than (+), binding region of the α9 subunit. In light of this unexpected discovery, we sought to determine other interacting residues with α-CTx RgIA. A previous molecular modeling study, based on the structure of the homologous molluscan acetylcholine-binding protein, predicted that RgIA interacts with three residues on the α9(+) face and two residues on the α10(-) face of the α9α10 nAChR. However, mutations of these residues had little or no effect on toxin block of the α9α10 nAChR. In contrast, mutations of homologous residues in the opposing nAChR subunits (α10 Ε197, P200 and α9 T61, D121) resulted in 19- to 1700-fold loss of toxin activity. Based on the crystal structure of the extracellular domain (ECD) of human α9 nAChR, we modeled the rat α9α10 ECD and its complexes with α-CTx RgIA and acetylcholine. Our data support the interaction of α-CTx RgIA at the α10/α9 rather than the α9/α10 nAChR subunit interface, and may facilitate the development of selective ligands with therapeutic potential.
Assuntos
Conotoxinas/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Dados de Sequência Molecular , Subunidades Proteicas/metabolismo , RatosRESUMO
Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) cooperate to trim antigenic peptide precursors for loading onto MHC class I molecules and help regulate the adaptive immune response. Common coding single nucleotide polymorphisms in ERAP1 and ERAP2 have been linked with predisposition to human diseases ranging from viral and bacterial infections to autoimmunity and cancer. It has been hypothesized that altered Ag processing by these enzymes is a causal link to disease etiology, but the molecular mechanisms are obscure. We report in this article that the common ERAP2 single nucleotide polymorphism rs2549782 that codes for amino acid variation N392K leads to alterations in both the activity and the specificity of the enzyme. Specifically, the 392N allele excises hydrophobic N-terminal residues from epitope precursors up to 165-fold faster compared with the 392K allele, although both alleles are very similar in excising positively charged N-terminal amino acids. These effects are primarily due to changes in the catalytic turnover rate (k(cat)) and not in the affinity for the substrate. X-ray crystallographic analysis of the ERAP2 392K allele suggests that the polymorphism interferes with the stabilization of the N terminus of the peptide both directly and indirectly through interactions with key residues participating in catalysis. This specificity switch allows the 392N allele of ERAP2 to supplement ERAP1 activity for the removal of hydrophobic N-terminal residues. Our results provide mechanistic insight to the association of this ERAP2 polymorphism with disease and support the idea that polymorphic variation in Ag processing enzymes constitutes a component of immune response variability in humans.
Assuntos
Aminopeptidases/genética , Apresentação de Antígeno/imunologia , Retículo Endoplasmático/enzimologia , Switching de Imunoglobulina/imunologia , Polimorfismo de Nucleotídeo Único/imunologia , Sequência de Aminoácidos , Apresentação de Antígeno/genética , Cristalografia por Raios X , Retículo Endoplasmático/genética , Retículo Endoplasmático/imunologia , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Variação Genética/imunologia , Células HeLa , Humanos , Switching de Imunoglobulina/genética , Dados de Sequência Molecular , Especificidade por Substrato/genética , Especificidade por Substrato/imunologiaRESUMO
Inhibition of Insulin-Regulated Aminopeptidase is being actively explored for the treatment of several human diseases and several classes of inhibitors have been developed although no clinical applications have been reported yet. Here, we combine enzymological analysis with x-ray crystallography to investigate the mechanism employed by two of the most studied inhibitors of IRAP, an aryl sulfonamide and a 2-amino-4H-benzopyran named HFI-419. Although both compounds have been hypothesized to target the enzyme's active site by competitive mechanisms, we discovered that they instead target previously unidentified proximal allosteric sites and utilize non-competitive inhibition mechanisms. X-ray crystallographic analysis demonstrated that the aryl sulfonamide stabilizes the closed, more active, conformation of the enzyme whereas HFI-419 locks the enzyme in a semi-open, and likely less active, conformation. HFI-419 potency is substrate-dependent and fails to effectively block the degradation of the physiological substrate cyclic peptide oxytocin. Our findings demonstrate alternative mechanisms for inhibiting IRAP through allosteric sites and conformational restricting and suggest that the pharmacology of HFI-419 may be more complicated than initially considered. Such conformation-specific interactions between IRAP and small molecules can be exploited for the design of more effective second-generation allosteric inhibitors.
Assuntos
Sítio Alostérico , Inibidores Enzimáticos , Insulina , Sulfonamidas , Humanos , Domínio Catalítico/efeitos dos fármacos , Cistinil Aminopeptidase/antagonistas & inibidores , Cistinil Aminopeptidase/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Insulina/metabolismo , Sulfonamidas/química , Sulfonamidas/farmacologia , Cristalografia por Raios X , Regulação Alostérica , Sítio Alostérico/efeitos dos fármacos , Células HEK293 , Células CHO , Animais , CricetulusRESUMO
Insulin-regulated aminopeptidase (IRAP) is an enzyme with important biological functions and the target of drug-discovery efforts. We combined in silico screening with a medicinal chemistry optimization campaign to discover a nanomolar inhibitor of IRAP based on a pyrazolylpyrimidine scaffold. This compound displays an excellent selectivity profile versus homologous aminopeptidases, and kinetic analysis suggests it utilizes an uncompetitive mechanism of action when inhibiting the cleavage of a typical dipeptidic substrate. Surprisingly, the compound is a poor inhibitor of the processing of the physiological cyclic peptide substrate oxytocin and a 10mer antigenic epitope precursor but displays a biphasic inhibition profile for the trimming of a 9mer antigenic peptide. While the compound reduces IRAP-dependent cross-presentation of an 8mer epitope in a cellular assay, it fails to block in vitro trimming of select epitope precursors. To gain insight into the mechanism and basis of this unusual selectivity for this inhibitor, we solved the crystal structure of its complex with IRAP. The structure indicated direct zinc(II) engagement by the pyrazolylpyrimidine scaffold and revealed that the compound binds to an open conformation of the enzyme in a pose that should block the conformational transition to the enzymatically active closed conformation previously observed for other low-molecular-weight inhibitors. This compound constitutes the first IRAP inhibitor targeting the active site that utilizes a conformation-specific mechanism of action, provides insight into the intricacies of the IRAP catalytic cycle, and highlights a novel approach to regulating IRAP activity by blocking its conformational rearrangements.
Assuntos
Cistinil Aminopeptidase , Cistinil Aminopeptidase/antagonistas & inibidores , Cistinil Aminopeptidase/química , Cistinil Aminopeptidase/metabolismo , Humanos , Cristalografia por Raios X , Especificidade por Substrato , Pirimidinas/química , Pirimidinas/farmacologia , Modelos Moleculares , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Conformação ProteicaRESUMO
BACKGROUND: Hypertension worsens outcomes in SARS-CoV-2 patients. Sartans, a type of antihypertensive angiotensin receptor blocker-(ARB), reduce COVID-19 morbidity and mortality by targeting angiotensin-converting enzyme-2 (ACE2). This study aimed to evaluate the antiviral and antihypertensive effects of nirmatrelvir, commercial sartans (candesartan, losartan, and losartan carboxylic (Exp3174)), and newly synthesized sartans (benzimidazole-N-biphenyl carboxyl (ACC519C) and benzimidazole-N-biphenyl tetrazole (ACC519T)), compared to nirmatrelvir, the antiviral component of Paxlovid. RESEARCH DESIGN AND METHODS: Surface plasmon resonance (SPR) and enzymatic studies assessed drug effects on ACE2. Antiviral abilities were tested with SARS-CoV-2-infected Vero E6 cells, and antihypertensive effects were evaluated using angiotensin II-contracted rabbit iliac arteries. RESULTS: Benzimidazole-based candesartan and ACC519C showed antiviral activity comparable to nirmatrelvir (95% inhibition). Imidazole-based losartan, Exp3174, and ACC519T were less potent (75%-80% and 50%, respectively), with Exp3174 being the least effective. SPR analysis indicated high sartans-ACE2 binding affinity. Candesartan and nirmatrelvir combined had greater inhibitory and cytopathic effects (3.96%) than individually (6.10% and 5.08%). ACE2 enzymatic assays showed varying effects of novel sartans on ACE2. ACC519T significantly reduced angiotensin II-mediated contraction, unlike nirmatrelvir and ACC519T(2). CONCLUSION: This study reports the discovery of a new class of benzimidazole-based sartans that significantly inhibit SARS-CoV-2, likely due to their interaction with ACE2.
Assuntos
Enzima de Conversão de Angiotensina 2 , Antivirais , Benzimidazóis , Tratamento Farmacológico da COVID-19 , SARS-CoV-2 , Benzimidazóis/farmacologia , Animais , Antivirais/farmacologia , Humanos , Chlorocebus aethiops , Enzima de Conversão de Angiotensina 2/metabolismo , SARS-CoV-2/efeitos dos fármacos , Células Vero , Coelhos , Antagonistas de Receptores de Angiotensina/farmacologia , Compostos de Bifenilo/farmacologia , Anti-Hipertensivos/farmacologia , Tetrazóis/farmacologia , Masculino , Hipertensão/tratamento farmacológico , COVID-19 , Losartan/farmacologia , Ressonância de Plasmônio de SuperfícieRESUMO
Endoplasmic reticulum aminopeptidase 2 (ERAP2) is an intracellular enzyme involved in the processing of antigenic peptides intended for presentation by major histocompatibility complex class I (MHCI) molecules. Because of its role in regulating immune responses, ERAP2 is an emerging pharmacological target. Phosphinic pseudopeptides are potent transition-state analogue inhibitors of ERAP2. Previous structure-activity studies have revealed a complex but ambiguous relationship between the occupation of putative specificity pockets and the inhibitor efficacy. To address these problems, we solved crystal structures of ERAP2 in complex with two phosphinic pseudotripeptide inhibitors. Both compounds are found in the catalytic site in a canonical orientation for transition-state analogues and utilize the S1 and S2' pockets in a similar fashion. Strikingly, their P1' side chains exhibit different orientations and make interactions with distinct shallow pockets near the ERAP2 active site. These structures suggest that S1' pocket usage in ERAP2 may be inhibitor-dependent and constitute useful starting templates for the further optimization of this class of compounds.
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[This corrects the article DOI: 10.3389/fphar.2019.00474.].
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The oxytocinase subfamily of M1 zinc aminopeptidases comprises emerging drug targets, including the ER-resident aminopeptidases 1 and 2 (ERAP1 and ERAP2) and insulin-regulated aminopeptidase (IRAP); however, reports on clinically relevant inhibitors are limited. Here we report a new synthetic approach of high diastereo- and regioselectivity for functionalization of the α-hydroxy-ß-amino acid scaffold of bestatin. Stereochemistry and mechanism of inhibition were investigated by a high-resolution X-ray crystal structure of ERAP1 in complex with a micromolar inhibitor. By exploring the P1 side-chain functionalities, we achieve significant potency and selectivity, and we report a cell-active, low-nanomolar inhibitor of IRAP with >120-fold selectivity over homologous enzymes. X-ray crystallographic analysis of IRAP in complex with this inhibitor suggest that interactions with the GAMEN loop is an unappreciated key determinant for potency and selectivity. Overall, our results suggest that α-hydroxy-ß-amino acid derivatives may constitute useful chemical tools and drug leads for this group of aminopeptidases.
Assuntos
Aminopeptidases , Insulina , Aminoácidos/farmacologia , Aminopeptidases/química , Cistinil Aminopeptidase , Leucina/análogos & derivadosRESUMO
Insulin-regulated aminopeptidase (IRAP) is a transmembrane zinc metallopeptidase with many important biological functions and an emerging pharmacological target. Although previous structural studies have given insight on how IRAP recognizes linear peptides, how it recognizes its physiological cyclic ligands remains elusive. Here, we report the first crystal structure of IRAP with the macrocyclic peptide inhibitor HA08 that combines structural elements from angiotensin IV and the physiological substrates oxytocin and vasopressin. The compound is found in the catalytic site in a near canonical substrate-like configuration and inhibits by a competitive mechanism. Comparison with previously solved structures of IRAP along with small-angle X-ray scattering experiments suggests that IRAP is in an open conformation in solution but undergoes a closing conformational change upon inhibitor binding. Stabilization of the closed conformation in combination with catalytic water exclusion by the tightly juxtaposed GAMEN loop is proposed as a mechanism of inhibition.
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The crystal structures of the C57A and V13G molecular variants of Allochromatium vinosum 2[4Fe-4S] ferredoxin (AlvinFd) and that of the homologous ferredoxin from Escherichia coli (EcFd) have been determined at 1.05-, 1.48-, and 1.65-A resolution, respectively. The present structures combined with cyclic voltammetry studies establish clear effects of the degree of exposure of the cluster with the lowest reduction potential (cluster I) towards less negative reduction potentials (E degrees ). This is better illustrated by V13G AlvinFd (high exposure, E degrees = -594 mV) and EcFd (low exposure, E degrees = -675 mV). In C57A AlvinFd, the movement of the protein backbone, as a result of replacing the noncoordinating Cys57 by Ala, leads to a +50-mV upshift of the potential of the nearby cluster I, by removal of polar interactions involving the thiolate group and adjustment of the hydrogen-bond network involving the cluster atoms. In addition, the present structures and other previously reported accurate structures of this family of ferredoxins indicate that polar interactions of side chains and water molecules with cluster II sulfur atoms, which are absent in the environment of cluster I, are correlated to the approximately 180-250 mV difference between the reduction potentials of clusters I and II. These findings provide insight into the significant effects of subtle structural differences of the protein and solvent environment around the clusters of [4Fe-4S] ferredoxins on their electrochemical properties.