Fig. 6: High-resolution WT T20S structure (2.1 Å) combine with ZYA-T20S structure shows αI12-αT13 play pivotal mechanistic role in switching the gate between open and closed states.

a View of WT T20S (yellow) IT switch residues, I12 and T13 (sticks) in the closed gate state. e Same as A but rotated 90°. i Same as e but rotated 45°. b, f, j | c, g, k | d, h, l Same as a, e, i but for ZYA-T20S (blue), T20S-αL81Y (pink), and PA26-T20S (green), respectively. m (Top) View of IT Switch (box) on a single α subunit, corresponding to a–d; (middle) same as top after being rotated 90 degrees as shown, corresponding to e–h; (bottom) same as middle after being rotated 90 degrees as shown, corresponding to I–l. n I12 (left residue) of the IT switch in proximity to V14 and A11 (sticks) from neighboring α subunit in the WT T20S, corresponding to closed gate state. o. Same view as n, except in ZYA-T20S in the open state. I12 does not interact with neighboring α residues but instead interacts under helix 0. p Lengthwise cross-section view of 20S, oriented as shown in n, o. q Overlay of WT T20S and ZYA-T20S showing the IT switch in the closed (WT T20S, yellow) and open (ZYA-T20S, blue) states. r 7 nM T20S (WT, I12A, I12F, or I12T) incubated with 44 nM PA26 or 15 nM PAN (supplemented with ATP and MgCl₂). LFP degradation rate (rfu/min) normalized as in Fig. 2e). s 7 nM T20S (WT, T13A, or T13I) incubated with 44 nM PA26 or 15 nM PAN (supplemented with ATP and MgCl₂). LFP degradation rate (rfu/min) normalized as in Fig. 2e. Data (means) are representative of three or more independent experiments each performed in triplicate. Error bars represent ± standard deviation.