Fig. 5
The length-dependent regulation of Ap4A on HAn formation. a EMSA was performed with 25 μM HINT1H114A or HINT1WT incubated with 700 μM AMP, Ap3A, Ap4A, Ap5A, and ATP separately. Only Ap4A could induce the HINT1 polymerization. The experiment was performed three times with similar results. Source data are provided as a Source Data file. b FRET assay showing that only Ap4A, but not Ap5A/Ap3A/AMP, could induce the interaction between HINT1V97D-CFP and HINT1V97D-YFP. FRET signal between CFP-YFP tandems was measured and set to 100% (error bars represent the SD of three experimental repeats). *P < 0.05, **P < 0.01, ***P < 0.001, from two-tailed Student’s t-test. Source data are provided as a Source Data file. c–f Negative stain EM of 10 μM HINT1WT incubated with Ap4A, Ap3A, AMP, and Ap5A separately. Only Ap4A could efficiently induce the HINT1 filament formation. Scale bars, 100 nm. g The conformation of ATP in the HINT1H114A-ATP complex structure. Only AMP portion was visible, suggesting the rest part was flexible or disordered. h The conformation of Ap3A in the HINT1H114A-Ap3A complex structure. Only adenosine moiety was visible. i The conformation of Ap5A in the HINT1H114A-Ap5A complex structure. Two adenosines of Ap5A fit in the two adenosine pockets in HINT1 dimer I and HINT1 dimer II separately. The five-phosphate linkage in Ap5A adopted a bent and zigzag conformation, comparing with the straight and tight Ap4A
