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. 2022 Jan 31;11:e74206. doi: 10.7554/eLife.74206

Figure 2. TIR multimerization and phase transition superactivates its intrinsic NAD + glycohydrolase activity.

(A) NADase activity of purified TIR at increasing TIR protein concentrations is shown. Activity was assessed by incubating TIR protein with 1 mM ε-NAD and monitoring the rate at which the fluorescent product ε-ADPR was produced. Curve represents a nonlinear regression fit of the NADase activity data points (n = 2). (B) NADase activity of 2.5 μM TIR incubated in the presence of 25% (w/v) of macro- (PEG 8000, PEG 3350, and dextran) and micro- (sucrose and glycerol) viscogens was assessed as described in A (n = 2). (C) Dose dependency of macroviscogens on the NADase activity of TIR is shown. A total of 2.5 μM TIR protein was incubated with the indicated PEG compounds at concentrations from 0% to 30% (w/v). NADase activity was assessed as described in A (n = 2). (D) Steady-state kinetic analysis of 2.5 µM TIR incubated in 0–30% (w/v) of PEG 3350 with the ε-NAD substrate at concentrations from 0 to 4000 μM was assessed as described in A. (n = 2). From the steady-state kinetic analysis performed in D, Km(E), kcat (F), and kcat/Km (G) were determined at each PEG 3350 concentration. (H) SDS-PAGE analysis of TIR protein fractions incubated with increasing concentrations of PEG 3350 precentrifugation (C) and after centrifugation, the soluble (S) and pellet (P) protein fractions. NADase activity of TIR protein in each fraction and at each concentration of PEG 3350 was assessed, as described in A, and is represented below the gel image (n = 2, representative image shown). (I) Steady-state kinetic analysis of TIR wild-type, oligomerization mutants (TIRG747P and TIRH833A), and catalytic mutants (TIRE788Q and TIRE788A) in 25% PEG 3350 with 0–2000 μM ε-NAD was assessed as described in D. The inset image outlined in red is an enlarged image of the mutant kinetic data. Kinetic parameters (Km, kcat, and kcat/Km) are shown in the table below the graph (n = 3). (J, K) SDS-PAGE analysis of TIR wild-type, oligomerization mutant (TIRG747P) and catalytic mutants (TIRE788Q and TIRE788A) precipitation in the presence of 25% PEG 3350. Gel represents the soluble (S) and pellet (P) protein fractions of wild-type and mutant TIR following incubation with PEG 3350 and centrifugation. TIRG747P and TIRE788Q were assessed with 10 μM protein in J, and TIRE788A was assessed with 3 μM protein in K (a lower concentration was used for TIRE788A assays because the yield of the purified TIRE788A mutant was low). Quantification of replicates represented below gel images (n = 4, representative images shown). *equals p < 0.05 by one-way ANOVA in J and unpaired t-test in K. (L) Effect of 1,6-hexanediol on TIR NADase activity is shown. TIR protein was incubated in the presence or absence of either 25% PEG 3350 or 500 mM citrate and treated with either 0 or 2% 1,6-hexanediol. The NADase activity of TIR for each condition was assessed using the ε-NAD substrate assay (n = 3). *equals p < 0.05 (unpaired t-test). (M) The NADase activity of TIR protein incubated with either 25% PEG 3350 or 500 mM citrate before (precentrifugation, n = 2) and after centrifugation, the supernatant (n = 4) and precipitant (n = 2) fractions. Precipitation fractions were resuspended in buffer alone or buffer containing 25% PEG 3350 or 500 mM citrate, and NADase activity was assessed. (N) Negative stain electron microscopy in either the absence or presence of 500 mM citrate (diameter of particles = 8.9 nm ± 1.2, n = 65). Representative circular particles are labeled with arrowheads. All error bars reflect SEM. See also Figure 2—figure supplements 1 and 2.

Figure 2—source data 1. Figure 2A NADase activity of purified TIR at increasing TIR protein concentrations.
Figure 2—source data 2. Figure 2B NADase activity of 2.5 μM TIR incubated in the presence of 25% (w/v) of macro- (PEG 8000, PEG 3350, and dextran) and micro- (sucrose and glycerol) viscogens.
Figure 2—source data 3. Figure 2C Dose dependency of macroviscogens on the NADase activity of TIR.
Figure 2—source data 4. Figure 2D Steady-state kinetic analysis of 2.5 μM TIR incubated in 0%–30% (w/v) of PEG 3350.
Figure 2—source data 5. Figure 2H NADase activity of TIR protein in each fraction and at each concentration of PEG 3350.
Figure 2—source data 6. Figure 2I Steady-state kinetic analysis of TIR wild-type, oligomerization mutant (TIRG747P, TIRH833A), and catalytic mutants (TIRE788Q and TIRE788A) in 25% PEG 3350.
Figure 2—source data 7. Figure 2J Quantification of SDS-PAGE analysis of TIR: wild-type, oligomerization mutant (TIRG747P) and catalytic mutant (TIRE788Q), precipitation in the presence of 25% PEG 3350.
Figure 2—source data 8. Figure 2K Quantification of SDS-PAGE analysis of TIR: wild-type and catalytic mutant (TIRE788A), precipitation in the presence of 25% PEG 3350.
Figure 2—source data 9. Figure 2L Effect of 1,6-hexanediol on TIR NADase activity.
Figure 2—source data 10. Figure 2M NADase activity of TIR protein incubated with either 25% PEG 3350 or 500 mM citrate before (precentrifugation) and after centrifugation, the supernatant and precipitant fractions.

Figure 2.

Figure 2—figure supplement 1. TIR multimerization and phase transition superactivates its intrinsic NAD + glycohydrolase activity.

Figure 2—figure supplement 1.

(A) Enzyme concentration dependence in the presence and absence of either 25 % PEG 3350 or 500 mM sodium citrate (n = 2). (B) Dose-dependence of TIR NADase activity on citrate concentration as described in Figure 2C, except using citrate (n = 2). (C) Steady-state kinetic analysis of TIR as described in Figure 2D, except in the presence of citrate (n = 2). (D–F) From the steady-state kinetic analysis performed in (C), Km(D), kcat (E), and kcat/Km (F) were determined at each citrate concentration. (G) Effect of citrate on TIR aggregation and associated activity as described in Figure 2H (n = 2, representative images shown). (H) Steady-state kinetic analysis of TIR mutants as described in Figure 2I, except in the presence of citrate (n = 3). (I, J) Precipitation of TIR mutants as described in Figure 2J and K, except in the presence of citrate (n = 4, representative images shown). See also Figure 2.
Figure 2—figure supplement 1—source data 1. 1A enzyme concentration dependence in presence and absence of either 25% PEG 3350 or 500 mM sodium citrate.
Figure 2—figure supplement 1—source data 2. 1B Dose dependency of citrate on the NADase activity of TIR.
Figure 2—figure supplement 1—source data 3. 1C Steady-state kinetic analysis of 2.5 μM TIR incubated in 0–1000 mM sodium citrate.
Figure 2—figure supplement 1—source data 4. 1G NADase activity of TIR protein in each fraction and at each concentration of citrate.
Figure 2—figure supplement 1—source data 5. 1H Steady-state kinetic analysis of TIR wild-type, oligomerization mutant (TIRG747P, TIRH833A), and catalytic mutants (TIRE788Q and TIRE788A) in 500 mM sodium citrate.
Figure 2—figure supplement 1—source data 6. 1I Quantification of SDS-PAGE analysis of TIR: wild-type, oligomerization mutant (TIRG747P) and catalytic mutant (TIRE788Q), precipitation in the presence of 500 mM sodium citrate.
Figure 2—figure supplement 1—source data 7. 1J Quantification of SDS-PAGE analysis of TIR: wild-type and catalytic mutant (TIRE788A), precipitation in the presence of 500 mM sodium citrate.
Figure 2—figure supplement 2. TIR multimerization and phase transition superactivates its intrinsic NAD + glycohydrolase activity.

Figure 2—figure supplement 2.

(A) SDS-PAGE analysis of TIR protein incubated first with 25% PEG 3350 or 500 mM citrate and subsequently with 2% 1,6-hexanediol; analysis was performed on TIR samples taken precentrifugation (C) or on the soluble (S) and pellet (P) fractions taken after centrifugation (n = 2, representative image shown). (B) SDS-PAGE analysis of TIR protein incubated first with 0 or 2% 1,6-hexanediol and subsequently with 25% PEG 3350 or 500 mM citrate (n = 2, representative image shown); analysis of protein fractions as in (A). (C) SDS-PAGE analysis of TIR protein in the soluble (S) and pellet (P) fractions following incubation with either PEG 3350 or citrate and centrifugation (one spin). Pellet fractions were subsequently resuspended in only buffer or buffer containing 25% PEG 3350 or 500 mM citrate and centrifuged a second time to isolate the soluble (S) and pellet (P) fractions (n = 2, representative image shown). (D) SDS-PAGE analysis of TIR in the soluble (S) and pellet (P) fractions incubated with or without 25% PEG 3350 at the indicated pH (n = 2, representative image shown). TIR steady-state kinetic parameters Km (E), kcat (F), kcat/Km (G) are shown at the indicated pH (n = 2). See also Figure 2.