DataSheet_1_Can ERAP1 and ERAP2 Form Functional Heterodimers? A Structural Dynamics Investigation.pdf

Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) play important roles in the generation of antigenic peptides presented by Major Histocompatibility Class I (MHCI) molecules and indirectly regulate adaptive immune responses. Although the discrete function of these enzymes has been extensively characterized, recent reports have suggested that they can also form heterodimers with functional consequences. However, lack of structural characterization of a putative ERAP1/ERAP2 dimer has limited our understanding of its biological role and significance. To address this, we employed computational molecular dynamics calculations to explore the topology of interactions between these two, based on experimentally determined homo-dimerization interfaces observed in crystal structures of ERAP2 or homologous enzymes. Our analysis of 8 possible dimerization models, suggested that the most likely ERAP1/ERAP2 heterodimerization topology involves the exon 10 loop, a non-conserved loop previously implicated in interactions between ERAP1 and the disulfide-bond shuffling chaperone ERp44. This dimerization topology allows access to the active site of both enzymes and is consistent with a previously reported construct in which ERAP1 and ERAP2 were linked by Fos/Jun zipper tags. The proposed model constitutes a tentative structural template to help understand the physiological role and significance of ERAP1/ERAP2 molecular interactions.</p

DataSheet_2_Can ERAP1 and ERAP2 Form Functional Heterodimers? A Structural Dynamics Investigation.zip

Endoplasmic reticulum aminopeptidases 1 and 2 (ERAP1 and ERAP2) play important roles in the generation of antigenic peptides presented by Major Histocompatibility Class I (MHCI) molecules and indirectly regulate adaptive immune responses. Although the discrete function of these enzymes has been extensively characterized, recent reports have suggested that they can also form heterodimers with functional consequences. However, lack of structural characterization of a putative ERAP1/ERAP2 dimer has limited our understanding of its biological role and significance. To address this, we employed computational molecular dynamics calculations to explore the topology of interactions between these two, based on experimentally determined homo-dimerization interfaces observed in crystal structures of ERAP2 or homologous enzymes. Our analysis of 8 possible dimerization models, suggested that the most likely ERAP1/ERAP2 heterodimerization topology involves the exon 10 loop, a non-conserved loop previously implicated in interactions between ERAP1 and the disulfide-bond shuffling chaperone ERp44. This dimerization topology allows access to the active site of both enzymes and is consistent with a previously reported construct in which ERAP1 and ERAP2 were linked by Fos/Jun zipper tags. The proposed model constitutes a tentative structural template to help understand the physiological role and significance of ERAP1/ERAP2 molecular interactions.</p

Screening Identifies Thimerosal as a Selective Inhibitor of Endoplasmic Reticulum Aminopeptidase 1

We employed virtual screening followed by <i>in vitro</i> evaluation to discover novel inhibitors of ER aminopeptidase 1, an important enzyme for the human adaptive immune response that has emerged as an attractive target for cancer immunotherapy and the control of autoimmunity. Screening hits included three structurally related compounds carrying the (<i>E</i>)-<i>N</i>′-((1<i>H</i>-indol-3-yl)­methylene)-1<i>H</i>-pyrazole-5-carbohydrazide scaffold and (2-carboxylatophenyl)­sulfanyl-ethylmercury as novel ERAP1 inhibitors. The latter, also known as thimerosal, a common component in vaccines, was found to inhibit ERAP1 in the submicromolar range and to present strong selectivity versus the homologous aminopeptidases ERAP2 and IRAP. Cell-based analysis indicated that thimerosal can effectively reduce ERAP1-dependent cross-presentation by dendritic cells in a dose-dependent manner

Comparative Experimental and Computational Study of Monoalkyl Chain Phosphatidylcholine-Containing Thermoresponsive Liposomes

Liposomes containing lysophospholipids are intensively studied as drug delivery systems that are stable at normal body temperature but exhibit fast release of their drug load at slightly elevated temperatures. In this study, the stability and release properties of dipalmitoylglycerophosphocholine (DPPC)-based liposomes incorporating the commonly used lysophosphatidylocholine (lyso-PC), and a series of monoalkyl chain ether-linked phosphatidylcholine, i.e., the biologically relevant monoalkyl chain platelet activating factor (PAF) and its derivatives lyso-PAF and methyl-PAF, were investigated. To this end a series of PEGylated small unilamellar liposomes with DPPC:monoalkyl lipid compositions of 5% and 10% molar ratio were prepared and compared with regard to stability (37 °C) and release properties at elevated temperatures (38–43 °C). All systems were characterized with respect to size distribution, ζ-potential, and phase transition characteristics. The presence of ether–lipids endows liposomes with superior (∼10% increase) release properties at 5% incorporation compared to lyso-PC, while at 10% molar ratio the formulations do not differ significantly, the release being close to 90%. The findings are supported by atomistic molecular dynamics simulations that suggest a correlation between the enhanced permeability and increased penetration of water molecules within the bilayers with density fluctuations resulting from the increased area-per-lipid and the disorder of the lysolipids alkyl chains

A Computational Approach to the Study of the Binding Mode of Dual ACE/NEP Inhibitors

Combined blockade of the renin−angiotensin−aldosterone system (RAAS) is an attractive therapeutic strategy for the treatment of cardiovascular diseases. Vasopeptidase inhibitors are a group of compounds capable of inhibiting more than one enzyme, which leads to potentiation of natriuretic peptide actions and suppression of the RAAS. In this study, molecular modeling has been used to elucidate key structural features that govern the binding and/or selectivity of a single compound toward the zinc catalytic sites of the N- and C-domains of the angiotensin-converting enzyme (ACE) and the neutral endopeptidase (NEP). Eleven dual inhibitors were categorized in three classes, according to their zinc binding groups. Analysis of their docked conformers revealed the molecular environment of the catalytic sites and the specific interactions between the inhibitors and amino acid residues that are important for selectivity and cooperativity. In addition, inhibitors were predicted to bind to the C-domain of the ACE with greater affinity than the N-domain, with an average difference in the free energy of binding ∼2−3 kcal mol−1. Residues that were identified to actively participate in the binding and stabilizating of the enzyme−inhibitor complexes were analyzed in a consensus way for both the ACE and the NEP. These atomic-level insights into enzyme−ligand binding can be used to drive new structure-based drug design processes in the quest for more selective and effective vasopeptidase inhibitors

Simulated Interactions between Angiotensin-Converting Enzyme and Substrate Gonadotropin-Releasing Hormone: Novel Insights into Domain Selectivity^†

Human angiotensin-I converting enzyme (ACE) is a central component of the renin-angiotensin system and a major target for cardiovascular therapies. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. On the basis of the recently determined crystal structures of both ACE domains, we have studied their complexes with gonadotropin-releasing hormone (GnRH), which is cleaved releasing both the protected NH2- and COOH-terminal tripeptides. This is the first molecular modeling study of an ACE−peptide substrate complex that examines the structural basis of ACE's endopeptidase activity and offers novel insights into subsites that are distant from the obligatory binding site and were not identified in the crystal structures. Our data indicate that a bridging interaction between Arg500 of the N-domain and Arg8 of GnRH that involves a buried chloride ion may account for its role in the specificity of the N-domain for endoproteolytic cleavage of the substrate at the NH2-terminus in vitro. In support of this, the protected NH2-terminal dipeptide of GnRH exhibits stronger interactions than the protected COOH-terminal dipeptide with the N-domain of ACE. Further comparison of the models of ACE−substrate complexes promotes our understanding of how the two domains differ in their function and specificity and provides an extension of the pharmacophore model used for structure-based drug design up to the S7 subsite of the enzyme

Correction to: LMTK3 inhibition affects microtubule stability

Correction to: Mol Cancer 20, 53 (2021) https://doi.org/10.1186/s12943-021-01345-3 Following the publication of the original article [1], the authors noticed errors on the figures introduced during the production step. Below are the errors: Fig. 2a: The labels for the y- and x-axes have been translocated upwards and need to be realigned with the axes. The x-axis should read: -Log2 (fold change). Fig. 2d: the quantification values in the NUSAP1 blots, for T47D and MDA-MB-231 cells, are no longer visible. The original article has been corrected

Data_Sheet_1_Crystal Structure of the Monomeric Extracellular Domain of α9 Nicotinic Receptor Subunit in Complex With α-Conotoxin RgIA: Molecular Dynamics Insights Into RgIA Binding to α9α10 Nicotinic Receptors.ZIP

The α9 subunit of nicotinic acetylcholine receptors (nAChRs) exists mainly in heteropentameric assemblies with α10. Accumulating data indicate the presence of three different binding sites in α9α10 nAChRs: the α9(+)/α9(−), the α9(+)/α10(−), and the α10(+)/α9(−). The major role of the principal (+) side of the extracellular domain (ECD) of α9 subunit in binding of the antagonists methyllylcaconitine and α-bungarotoxin was shown previously by the crystal structures of the monomeric α9-ECD with these molecules. Here we present the 2.26-Å resolution crystal structure of α9-ECD in complex with α-conotoxin (α-Ctx) RgIA, a potential drug for chronic pain, the first structure reported for a complex between an nAChR domain and an α-Ctx. Superposition of this structure with those of other α-Ctxs bound to the homologous pentameric acetylcholine binding proteins revealed significant similarities in the orientation of bound conotoxins, despite the monomeric state of the α9-ECD. In addition, ligand-binding studies calculated a binding affinity of RgIA to the α9-ECD at the low micromolar range. Given the high identity between α9 and α10 ECDs, particularly at their (+) sides, the presented structure was used as template for molecular dynamics simulations of the ECDs of the human α9α10 nAChR in pentameric assemblies. Our results support a favorable binding of RgIA at α9(+)/α9(−) or α10(+)/α9(−) rather than the α9(+)/α10(−) interface, in accordance with previous mutational and functional data.</p

Data_Sheet_2_Crystal Structure of the Monomeric Extracellular Domain of α9 Nicotinic Receptor Subunit in Complex With α-Conotoxin RgIA: Molecular Dynamics Insights Into RgIA Binding to α9α10 Nicotinic Receptors.docx

The α9 subunit of nicotinic acetylcholine receptors (nAChRs) exists mainly in heteropentameric assemblies with α10. Accumulating data indicate the presence of three different binding sites in α9α10 nAChRs: the α9(+)/α9(−), the α9(+)/α10(−), and the α10(+)/α9(−). The major role of the principal (+) side of the extracellular domain (ECD) of α9 subunit in binding of the antagonists methyllylcaconitine and α-bungarotoxin was shown previously by the crystal structures of the monomeric α9-ECD with these molecules. Here we present the 2.26-Å resolution crystal structure of α9-ECD in complex with α-conotoxin (α-Ctx) RgIA, a potential drug for chronic pain, the first structure reported for a complex between an nAChR domain and an α-Ctx. Superposition of this structure with those of other α-Ctxs bound to the homologous pentameric acetylcholine binding proteins revealed significant similarities in the orientation of bound conotoxins, despite the monomeric state of the α9-ECD. In addition, ligand-binding studies calculated a binding affinity of RgIA to the α9-ECD at the low micromolar range. Given the high identity between α9 and α10 ECDs, particularly at their (+) sides, the presented structure was used as template for molecular dynamics simulations of the ECDs of the human α9α10 nAChR in pentameric assemblies. Our results support a favorable binding of RgIA at α9(+)/α9(−) or α10(+)/α9(−) rather than the α9(+)/α10(−) interface, in accordance with previous mutational and functional data.</p

LMTK3 inhibition affects microtubule stability

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