Fig. 4: Dimerisation and functional investigation.
From: Full-length in meso structure and mechanism of rat kynurenine 3-monooxygenase inhibition
A, B Inter-β-sheet hydrogen bonds observed in domain I of Rat-KMO. The crystallographic dimer has 10 hydrogen bonds, in addition to intra hydrogen bonds, linking the dimers. Sticks and secondary structures are coloured using the same scheme as that in Fig. 1. C Dimerisation assays for Hs-KMO using NanoBRET. BRET units of non-GST-tagged Hs-KMO and its mutant were measured in HEK293T cells. Mutations at the dimer interface result in significantly lower BRET signals. Error bars represent standard deviation (n = 6, *p < 0.0001, two-tailed unpaired Student’s t-test). D Functional assay of Rat-KMO mutants in mitochondrial membranes. All mutants decreased catalytic activity of Rat-KMO, with activity being almost completely abolished in the presence of the β-sheet-disrupting Y185P mutation. For each sample, the enzyme concentration dilution factor (x-axis) is normalised to the intensity of anti-GST from western blot analyses.
