c21ebe9d27d2262d1b5c6bc2cab78f0d IntJouMolSci_a2021v2n6p3285iENG.pdf 566570537d9154f53f86bd3b95732d72d7f0c482 IntJouMolSci_a2021v2n6p3285iENG.pdf 538f5f33420566b12350791773a498bdf393eb992884122f9f6c70143a2d7868 IntJouMolSci_a2021v2n6p3285iENG.pdf Title: Conformational Heterogeneity and Cooperative Effects of Mammalian ALOX15 Subject: Arachidonic acid lipoxygenases (ALOXs) have been suggested to function as monomeric enzymes, but more recent data on rabbit ALOX15 indicated that there is a dynamic monomer-dimer equilibrium in aqueous solution. In the presence of an active site ligand (the ALOX15 inhibitor RS7) rabbit ALOX15 was crystalized as heterodimer and the X-ray coordinates of the two monomers within the dimer exhibit subtle structural differences. Using native polyacrylamide electrophoresis, we here observed that highly purified and predominantly monomeric rabbit ALOX15 and human ALOX15B are present in two conformers with distinct electrophoretic mobilities. In silico docking studies, molecular dynamics simulations, site directed mutagenesis experiments and kinetic measurements suggested that in aqueous solutions the two enzymes exhibit motional flexibility, which may impact the enzymatic properties. Keywords: lipoxygenases; crystal structure; protein–protein interactions; cooperative effects; molecular dynamics Author: Igor Ivanov, Alejandro Cruz, Alexander Zhuravlev, Almerinda Di Venere, Eleonora Nicolai, Sabine Stehling, José M. Lluch, Àngels González-Lafont and Hartmut Kuhn Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Mar 31 16:49:49 2021 CEST ModDate: Thu May 27 16:20:20 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 33088626 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- SBMXUO+VnURWPalladioL Type 1 Custom yes yes no 10 0 QCRZYJ+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 DGQQZW+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 YGBAWR+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 EPTQDJ+VnURWPalladioL-Bold Type 1 Custom yes yes no 28 0 IRWHZX+TimesNewRomanPSMT Type 1C WinAnsi yes yes no 36 0 CKUPDE+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 39 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 61 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 65 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes no 69 0 VVSYYB+EURM10 Type 1 Builtin yes yes no 81 0 RYQVMQ+CMSY10 Type 1 Builtin yes yes no 99 0 XIRVNA+EURB10 Type 1 Builtin yes yes no 131 0 WWNSGS+URWPalladioL-BoldItal Type 1 Custom yes yes no 135 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-04-01 02:33:53 CEST RepresentationInformation: IntJouMolSci_a2021v2n6p3285iENG.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2023-03-31 11:58:38 CEST Size: 33088626 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 431 FreeObjects: 9 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Structural Characterization of ALOX15 Isoenzymes by Native PAGE Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Consequences of N-terminal Truncation and Point Mutations of His585 on Conformational Heterogeneity of Rabbit ALOX15 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Impact of Point Mutations of Amino Acids Localized at the Inter-Monomer Contact Site on the Reaction Kinetics of Rabbit ALOX15 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: MD Simulation of the Structural Alterations of Rabbit ALOX15 Induced by Trp181Glu Exchange Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: AA and LA Binding Modes Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Analysis of Pre-Catalytic Structures Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Children: Item: Title: Conformational Heterogeneity of ALOX15 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Cooperativity of Rabbit ALOX15 Monomers during Lipoxygenase Reaction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Children: Item: Title: Chemicals Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Expression of Rabbit ALOX15 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: Expression of Human ALOX15B Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Item: Title: Purification of Rabbit ALOX15 and Human ALOX15B Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@63440df3 Item: Title: Estimation of the Hydrodynamic Radii for Wild-Type and Mutant ALOX15 Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@3aeaafa6 Item: Title: Native PAGE Electrophoresis Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@76a3e297 Item: Title: CD Measurements Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4d3167f4 Item: Title: Guanidine Denaturation Studies. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@ed9d034 Item: Title: Rabbit ALOX15 Activity Assay (Purified Enzyme) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@6121c9d6 Item: Title: Molecular Docking Simulations Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@87f383f Item: Title: Molecular Dynamics (MD) Simulations Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Info: Title: Conformational Heterogeneity and Cooperative Effects of Mammalian ALOX15 Author: Igor Ivanov, Alejandro Cruz, Alexander Zhuravlev, Almerinda Di Venere, Eleonora Nicolai, Sabine Stehling, José M. Lluch, Àngels González-Lafont and Hartmut Kuhn Subject: Arachidonic acid lipoxygenases (ALOXs) have been suggested to function as monomeric enzymes, but more recent data on rabbit ALOX15 indicated that there is a dynamic monomer-dimer equilibrium in aqueous solution. In the presence of an active site ligand (the ALOX15 inhibitor RS7) rabbit ALOX15 was crystalized as heterodimer and the X-ray coordinates of the two monomers within the dimer exhibit subtle structural differences. Using native polyacrylamide electrophoresis, we here observed that highly purified and predominantly monomeric rabbit ALOX15 and human ALOX15B are present in two conformers with distinct electrophoretic mobilities. In silico docking studies, molecular dynamics simulations, site directed mutagenesis experiments and kinetic measurements suggested that in aqueous solutions the two enzymes exhibit motional flexibility, which may impact the enzymatic properties. 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