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 *Corresponding author. E-mail address: ewa.skrzypczak-jankun@utoledo.edu. Present address: A. M. Aleem, Biochemistry Department, College of Sciences, King Saud University, Riyadh 11451, Kingdom of Saudi Arabia.Abbreviations used: LOX, lipoxygenase; r-12/15LOX, rabbit 12/15LOX; h-5LOX, human 5LOX; SAXS, small-angle X-ray scattering; hp-12LOX, human platelet-type 12S-LOX; PDB, Protein Data Bank. doi:10.1016/j.jmb.2011.04.035 J. Mol. Biol. (2011

Dimerization of human 5-lipoxygenase

“…3o8y shows a crystallographic dimer, which is not a thermodynamically stable complex (Shang et al , 2011 ). Therefore, this assembly might solely rise from crystal-packing.…”

“…In most reviews, 5-LO is mentioned as a monomeric enzyme (Rouzer and Samuelsson , 1985 ;R å dmark, 2002 ), nevertheless, it was previously shown that 5-LO from rat basophilic leukemia cells (RBL-1) forms dimers in the presence of calcium (Parker and Aykent , 1982 ). Recently, Shang et al analyzed dimerization and fl exibility of rabbit 12/15-LO and human 12-LO by small angle X-ray scattering (SAXS) (Shang et al , 2011 ). The outcome of their study was that (i) r12/15-LO is mainly monomeric, but dimerizes at higher protein concentrations and with increasing protein fl exibility, and (ii) that human 12-LO is stable as a dimer confi rming their former studies for 12-LO (Aleem et al, 2008).…”

Catalytic Multiplicity of 15-Lipoxygenase-1 Orthologs (ALOX15) of Different Species

“…A number of spectroscopic studies, such as small angle X-ray scattering [116,117], dynamic fluorescence studies and fluorescence resonance energy transfer measurements [44,118], as well as various computational methods [117,119] have been applied to explore the motional flexibility of rabALOX15 in aqueous solutions. Summarizing these results one may conclude that three elements contribute to structural flexibility: (1) Ligand binding induces conformational alterations: Binding of an inhibitor (R7) at the active site induces conformational alterations and helices α2-and α18 are particularly affected [112].…”

Estudo da estabilidade térmica da hemoglobina extracelular gigante de Glossoscolex paulistus (HbGp): efeitos do estado de oxidação do ferro do grupo heme, pH e presença de surfactante

“…As curvas p(r) experimentais foram obtidas usando o programa GNOM. Os ajustes mostrados junto com a p(r) experimental foram realizados com a combinação linear das curvas de p(r) teóricas obtidas para a estrutura cristalográfica da HbLt, mostradas na Figura Vários estudos, baseados na técnica de SAXS, são reportados na literatura para os processos de dissociação, desnaturação e agregação para diferentes proteínas, induzidos por variação de pH, altas concentrações de sal e presença de cloreto de guanidina [44,45,49,[61][62][63][64][65][66].…”

“…Nesses estudos a caracterização de espécies dissociadas em solução também é descrita, principalmente para sistemas envolvendo o equilíbrio de homo-dimeros com os monômeros correspondentes [62,64]. Além disso, existem também estudos para proteínas oligoméricas, tais como, homo-tetrâmero e homo-hexâmero [58,66].…”