Figure 6. The Arg29_VHH–Glu67_RTA interaction is predicted to be both necessary and sufficient to enhance RTA binding affinity.

(Panel A) Distributions of V_HH–RTA interaction energy over ensembles of 5,000 computational models for V1C7, V5C1, and respective single-point mutants at V_HH position 29, named V1C7_G29R and V5C1_R29G. The Gly29Arg mutation in V1C7_G29R causes an average 2.5-REU shift to more favorable interaction energies, which become more similar to those of V5C1. Conversely, because of the Arg29Gly mutation, the interaction energies of V5C1_R29G shift by 2.4 REU toward less favorable values, approximating more closely those of V1C7. (Panel B) Distributions of X29_VHH–Glu67_RTA interaction energy over the ensembles of models; X = Gly for V1C7 and V5C1_R29G, X = Arg for V5C1 and V1C7_G29R. The Arg29_VHH–Glu67_RTA interaction of V1C7_G29R is as favorable as that of V5C1 (−3.1 and −3.2 REUs on average, respectively). Such an interaction is favorable enough to explain V1C7_G29R’s enhancement in V_HH–RTA interaction energy (−2.5 REUs on average) and, therefore, the prediction that V1C7_G29R and V5C1 have similar RTA binding affinities. On the other hand, absence of the Gly29_VHH–Glu67_RTA interaction (μ = 0.0) can explain, for the most part, why V1C7 and V5C1_R29G are predicted to bind RTA similarly weakly.