Figure 4. Dissection of the interactions between MD2 and MraY_AA elucidates the chemical logic of MraY_AA inhibition.

a, Composite simulated annealing 2F_o–F_c omit electron density of MD2 in the crystal structure of MD2-bound MraY_AA at 1.7σ. The TMs are colored as in Fig. 3, a-b. The residues forming side chain interactions with MD2 are labeled. b, A two-dimensional representation of the interactions between MD2 and MraY_AA. Hydrogen bonds (3.2 Å cutoff) are indicated with black dashed lines and π–π contacts are indicated with red dashes. Mutation of residues with red colored labels resulted in a larger than five-fold increase in the K_D of MD2 and those with blue residue labels are nearly inactive. c, Representative ITC raw data and binding isotherm for MD2 titrated into MraY_AA in the absence of added Mg²⁺; K_D = 14.8 nM, ΔH° = −8.3 kcal/mol. A similar K_D is observed for MD2 titrated into MraY_AA with added Mg²⁺. d, Representative ITC raw data and binding isotherm for 5-aminoribosyl-3-deoxy uridine titrated into MraY_AA WT; K_D = 283 nM, ΔH° = −16.4 kcal/mol. Each ITC experiment was performed in triplicate (technical replicates) and mean thermodynamic parameters are shown in Extended Data Table 2.