title: Probing dimerization and structural flexibility of mammalian lipoxygenases by small-angle X-ray scattering.
abstract: Human lipoxygenases (LOXs) and their metabolites have a great impact on human homeostasis and are of interest for targeted drug design. This goal requires detailed knowledge of their structures and an understanding of structure-function relationship. At the moment, there are two complete crystal structures for mammalian LOX [rabbit 12/15LOX (r-12/15LOX) and human 5LOX (h-5LOX)] and a fragment of human 12LOX. The low-resolution structures in solution for various LOX isoforms have brought about controversial results. Here we explored the behavior of r-12/15LOX in aqueous solution under different conditions (salt and pH) by small-angle X-ray scattering (SAXS) and compared it with human platelet-type 12S-LOX (hp-12LOX) and h-5LOX. Thermodynamic calculations concerning the stability of molecular assemblies, thermal motion analysis [TLSMD (translation, libration, and screw rotation motion detection based on crystallographic temperature factor B(j))], and results of SAXS analyses brought about the following conclusions: (i) in contrast to its crystal structure, r-12/15LOX functions as a monomer that dominates in solution; (ii) it dimerizes at higher protein concentrations in the presence of salt and with increasing degree of motional freedom of the N-terminal PLAT domain, as suggested by the Y98,614→R double mutant; (iii) in aqueous solutions, hp-12LOX is stable as a dimer, in contrast to h-5LOX and r-12/15LOX, which are monomeric; and (iv) all three mammalian isozymes show a high level of flexibility not only for the PLAT domain but also for other subdomains of the catalytic part in TLS (translation, libration, and screw rotation) analysis and hp-12LOX in SAXS.
authors.affiliation: European Molecular Biology Laboratory, Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany.
authors.forename: Weifeng
authors.initials: W
authors.lastname: Shang
authors.forename: Igor
authors.initials: I
authors.lastname: Ivanov
authors.forename: Dmitri I
authors.initials: DI
authors.lastname: Svergun
authors.forename: Oleg Y
authors.initials: OY
authors.lastname: Borbulevych
authors.forename: Ansari M
authors.initials: AM
authors.lastname: Aleem
authors.forename: Sabine
authors.initials: S
authors.lastname: Stehling
authors.forename: Jerzy
authors.initials: J
authors.lastname: Jankun
authors.forename: Hartmut
authors.initials: H
authors.lastname: Kühn
authors.forename: Ewa
authors.initials: E
authors.lastname: Skrzypczak-Jankun
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006706:Homeostasis; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D058945:Lipoxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012492:Salts / Q000737:chemistry; D053838:Scattering, Small Angle; D012996:Solutions / Q000737:chemistry; D013329:Structure-Activity Relationship; D013816:Thermodynamics
chemical_list: D007527:Isoenzymes; D012492:Salts; D012996:Solutions; D058945:Lipoxygenases