Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 409, Issue 4, Pages 654-668Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2011.04.035
Keywords
mammalian lipoxygenase; small-angle X-ray scattering; thermodynamic stability; thermal motion analysis; structure-function relationship
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Funding
- DFG [GRK 1673]
- European Molecular Biology Laboratory, Hamburg Outstation [SAXS-09-036, SAXS-10-004]
- European Commission
- Frederick M. Douglass Foundation
- Frank D. Stranaham Endowment Fund for Oncological Research
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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, ibration, 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. (C) 2011 Elsevier Ltd. All rights reserved.
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