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. 2018 Nov 27;7:e41604. doi: 10.7554/eLife.41604

Figure 4. Characterization of activator binding site mutants.

(a) Perturbation of the activator binding site leads to loss of responsiveness to compound 1, although the G71I and Y51S/G71I variants are themselves stabilized compared to WT LCAT. LPLA2 variants, however, do not bind to 1, and chimeric swaps are destabilized. Data are mean ± s.e.m. of at least three independent experiments performed in duplicate. ****p<0.0001 by one-way analysis of variance followed by Tukey’s multiple comparisons post-test. Each protein without ligand was compared to that same variant with compound 1, and WT LCAT was compared to each LCAT variant. Non-significant comparisons are not shown. (b) Plot used to determine kon, koff, and hence Kd from BLI data for LCAT binding to HDL. Data are mean ± s.e.m. of three independent experiments. (c) DHE acyltransferase assay with peptide HDLs comparing the absence (solid lines) and presence (dashed lines) of 5 μM compound 2, which was used in this assay instead of 1 due to its lower background fluorescence. Data are mean ± s.e.m. of three independent experiments performed in triplicate. (d) Titration of compound 1 in the MUP hydrolysis assay. Data were normalized to basal activity of 100% for each variant to give percent activation. Data are mean ± s.e.m. of three independent experiments. (e) Titration of compound 2 in the DHE acyltransferase assay. Data are mean ± s.e.m. of three independent experiments performed in triplicate.

Figure 4.

Figure 4—figure supplement 1. Biochemical characterization of LCAT variants.

Figure 4—figure supplement 1.

(a) SDS-PAGE gels of purified LCAT variants. Left panel shows representative variants used in pNPB, MUP, and DSF experiments that were polished via Superdex 75. Right panel shows representative variants used for DHE acyltransferase and BLI assays. Approximately 1.5 µg of each purified LCAT variant was loaded in each lane. (b) Rates of pNPB hydrolysis for LCAT variants. Data are mean ± s.e.m. of at least three independent experiments. ***p=0.0004, ****p<0.0001 by one-way analysis of variance followed by Tukey’s multiple comparisons post-test. Each protein without ligand was compared to that same variant with ligand, and WT LCAT was compared to each LCAT variant. Non-significant comparisons are not shown. (c) Comparison of basal MUP hydrolysis. Data are mean ± s.e.m. of three independent experiments performed with ≥27 repeats. **p=0.0070 by a two-tailed unpaired t test. WT was compared to each LCAT variant and non-significant comparisons are not shown.
Figure 4—figure supplement 2. Representative BLI data for LCAT-Arg244 variants.

Figure 4—figure supplement 2.

(a) LCAT analyzed with ApoA-I HDLs at different concentrations in order to determine Kd. R244A is in black to gray, whereas R244A with compound 1 is in navy to light blue. (b) Same as in (a) but with R244H. (c) Plot used to determine kon (slope of fit line), koff (y-intercept), and hence Kd for LCAT binding to HDL. Data are mean ± s.e.m. of three independent experiments. (d) – (f) Individual Kd (d), koff (e), and kon (f) values were calculated for each experiment. Data are mean ± s.e.m. of three independent experiments. * 0.01 <P < 0.05, ** 0.001 <P < 0.01, *** 0.0001 < P < 0.001, ****p<0.0001 by one-way analysis of variance followed by Tukey’s multiple comparisons post-test. Each protein without ligand was compared to that same variant with 1, and WT LCAT was compared to each dataset. R244A was also compared to R244H. Non-significant comparisons are not shown.