Fig. 2: Sequential conformational transitions of HtpG induced by ATP binding.

a Overlay of Methyl-TROSY spectra of apo (gray) and AMP-PNP-bound (green) HtpG, highlighting labeled residues exhibiting resonance splitting. b Plot of chemical shift perturbations (CSPs) across residue numbers for AMP-PNP-bound HtpG, with negative bars indicating doublet and triplet splitting signals (rosy and purple, respectively). c Schematic representation of the sequential conformational transition pathway of HtpG from the open to the closed state upon ATP binding. d Superposition of HtpG structures in open (gray), NTD-rotated (light pink), and closed (green) states, with splitting profiles for selected residues depicted as red spheres. Dashed arrows indicate linear shifts associated with triplet splitting. e Overlay of Methyl-TROSY spectra of apo (gray), AMP-PNP-bound (green) HtpG, and HtpG^NM in AMP-PNP-bound state (purple) for residues L278 and L233. f Population distribution analysis for the second conformational state in AMP-PNP-bound HtpG^FL (blue) and HtpG^NM (black). Data are presented as individual data points, with mean ± SEM, based on n = 10 biological replicates for each group (ten representative residues selected; refer to Methods for details). Statistical significance was assessed using a two-sided t-test, with p = 0.0004 as indicated by asterisks. g Negative stain EM analysis of AMP-PNP-bound HtpG, with additional images available in Fig. S4. h Scheme of the rugged energy landscape for the polymorphic AMP-PNP-bound HtpG. i Structural visualization based on PDB 2CG9, illustrating the role of residue R33 in the active site interaction network. j Overlay of Methyl-TROSY spectra for apo (gray), AMP-PNP-bound (green) HtpG, and the AMP-PNP-bound HtpG^R33A mutant (purple), showing residues representative of the mutation-induced lack of resonance splitting. k Comparative analysis of turnover rates between WT-HtpG and the HtpG^R33A mutant. Data from three replicates (n = 3) are presented as the mean ± SEM.