(a) Scheme for quantifying the cooperativity of interactions of a specific side chain. The stability changes (ΔΔGoU) induced by the same mutation (black star) were probed with two biotin pairs, 95/172N-BtnPyr2 and 172/267C-BtnPyr2 located in the N- and C-terminal regions, respectively, and compared to each other to yield ΔΔΔGoU using equation (1). The cyan-backbone region designates subdomain I (TM1-L1-TM2-TM3-L3198), which ends at residue 198 in the L3 loop (marked with a magenta wedge) and the yellow-backbone region (L3199-TM4-TM5-L5-TM6) indicates subdomain II. The uncertainty of the subdomain-division point was ±20–30 residues around residue 198. The detailed strategy for the subdomain dissection and the estimation of the uncertainty is described in Supplementary Fig. 10. Catalytic dyad composed of Ser201/His254 is shown as spheres. (b) Cooperativity map at a side-chain resolution. The map shows the “cooperative” (green, |ΔΔΔGoU|≤RT=0.6 kcal/mol) and “localized” (|ΔΔΔGoU|>RT) side-chain interactions. Localized interactions were further divided using additional cut-off energy values, 2RT≥|ΔΔΔGoU|>RT (“moderately-localized” interactions) and |ΔΔΔGoU|>2RT (“highly-localized” interactions). Each side chain was color-coded based on these criteria for ΔΔΔGoU as shown in the figure. Interactions mediated by residues G261 and A265 (denoted with stars) were “over-propagated”. Errors in individual ΔGoU were ±0.1−±0.2 kcal/mol (mean ± s. d. from fitting) and errors in ΔΔΔGoU ranged from ±0.1−±0.4 kcal/mol, which were calculated using the propagation of errors in ΔGoU (Supplementary Table 1).