Fig. 4: Wild-type NicA2 and mutant v321 populate distinct conformational landscapes.

a, The crystal structure of wild-type NicA2 (PDB ID 5TTJ) is displayed with a tunnel ~1.4 Å in diameter identified by CAVER simulation rendered in magenta. FAD is rendered in yellow, Y342 is rendered in gray, and labeled frequently mutated residues are rendered in orange (F104), purple (A107), green (D130), brown (H368) and turquoise (N462). b, Differential HDX-MS data are shown on the structure of wild-type NicA2 under either a ligand-free or NMM-bound condition. Sections of the protein backbone that are highlighted in red demonstrate greater solvent exchange in v321 than in the wild-type protein. Interestingly, we also observed other areas of deprotection in NicA2 away from this tunnel region, suggesting that some additional structural rearrangements are also occurring. Residues that are mutated in NicA2 v321 are rendered as black spheres. c, ¹⁹F NMR spectrum of wild-type NicA2 with the Y342tfmF substitution. The black trace represents the raw data, the colored curves represent fits deconvoluted using decon1d⁴⁴, and the gray trace represents residuals from the fit. d, ¹⁹F NMR spectrum of NicA2 v321 with the Y342tfmF substitution. e, PRE ratios for wild-type NicA2 and v321 in both apo- and NMM-bound states. A lower PRE ratio indicates increased accessibility to solvent. The mean is plotted with error bars that represent the s.d. of three replicates. Data were analyzed by one-way analysis of variance with a Tukey multiple comparisons post hoc test.

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