Mutagenesis, Hydrogen–Deuterium Exchange, and Molecular Docking Investigations Establish the Dimeric Interface of Human Platelet-Type 12-Lipoxygenase

Abstract

It was previously shown that human platelet 12Slipoxygenase (h12-LOX) exists as a dimer; however, the specific structure is unknown. In this study, we create a model of the dimer through a combination of computational methods, experimental mutagenesis, and hydrogen−deuterium exchange (HDX) investigations. Initially, Leu183 and Leu187 were replaced by negatively charged glutamate residues and neighboring aromatic residues were replaced with alanine residues (F174A/W176A/L183E/L187E/ Y191A). This quintuple mutant disrupted both the hydrophobic and π−π interactions, generating an h12-LOX monomer. To refine the determinants for dimer formation further, the L183E/L187E mutant was generated and the equilibrium shifted mostly toward the monomer. We then submitted the predicted monomeric structure to protein−protein docking to create a model of the dimeric complex. A total of nine of the top 10 most energetically favorable docking conformations predict a TOP-to-TOP dimeric arrangement of h12-LOX, with the α-helices containing a Leu-rich region (L172, L183, L187, and L194), corroborating our experimental results showing the importance of these hydrophobic interactions for dimerization. This model was supported by HDX investigations that demonstrated the stabilization of four, nonoverlapping peptides within helix α2 of the TOP subdomain for wt-h12-LOX, consistent with the dimer interface. Most importantly, our data reveal that the dimer and monomer of h12-LOX have distinct biochemical properties, suggesting that the structural changes due to dimerization have allosteric effects on active site catalysis and inhibitor binding.

N-Substituted 5-(1H-Indol-2-yl)-2-methoxyanilines Are Allosteric Inhibitors of the Linoleate Oxygenase Activity of Selected Mammalian ALOX15 Orthologs: Mechanism of Action

“…Mutagenesis studies suggested that rearrangements of intramolecular contacts of helix 18 may correlate with the volume of the putative substrate binding cavity of the catalytic monomer in the ligand-bound ALOX15 dimer and, thus, may fine-tune the reaction specificity of the enzyme . This hypothesis is consistent with the recent report showing that dimerization of human ALOX12 affects the enzymatic activity of this enzyme via allosteric mechanisms …”

“…42 This hypothesis is consistent with the recent report showing that dimerization of human ALOX12 affects the enzymatic activity of this enzyme via allosteric mechanisms. 53 Here, we report that the N-substituted 5-(1H-indol-2-yl)-2methoxyanilines 6b, 6c, 8b, and 8c may selectively inhibit linoleate oxygenase activity of rabbit and human ALOX15 with IC 50 values in the lower two-digit nanomolar range without affecting the rate of AA oxygenation. Since ALOX15 exhibits comparable binding affinities for LA and AA, 54,55 a conventional mode of inhibition predicts that the oxygenation of the two substrates should be inhibited to a similar extent at a given inhibitor concentration.…”

Thermodynamic and biophysical study of fatty acid effector binding to soybean lipoxygenase: implications for allostery driven by helix α2 dynamics

“…These biophysical data rule out oligomerization as the mechanism for allosteric modulation of SLO catalysis. This finding for the model plant 15-LOX contrasts the studies of mammalian 12/15-LOXs that have implicated dimerization for allosteric regulation [24,58]. Further, thermodynamic analysis of SLO from DSC experiments also excludes the possibility of a large conformational transition to SLO upon OS binding.…”

“…While these changes in HDX rates were interpreted in the context of protein dynamics, these HDX results alone do not establish a defined mechanism for SLO allostery. We have recently reported similarly altered HDX properties linked to dimerization of the human 12-LOX, when the native dimeric and mutant monomeric forms of the isozyme were compared [24]. These oligomerization changes in human 12-LOX also influenced substrate binding and reactivity, and the affinity for an isozyme selective inhibitor.…”

Biochemical and hydrogen-deuterium exchange studies of the single nucleotide polymorphism Y649C in human platelet 12-lipoxygenase linked to a bleeding disorder

“…Deuterated, pepsin-digested samples of h12-LOX were analyzed using a 1200 series LC system (Agilent, Santa Clara, CA) that was connected in-line with the LTQ Orbitrap XL mass spectrometer (Thermo), as described by our laboratory previously [ 19 , 29 ]. Mass spectral data corresponding to the HDX measurements were analyzed using HDX Workbench [ 30 ].…”