Fig. 8
Mutational studies. a Mapping of mutated residues on the structure. The side chains of mutated residues are shown in sticks and semi-transparent spheres for TtSlyDFL:S2 molecule A and colored according to activity relative to that of the wild type (see also Table 4): Dark red, 0–30 %; pink, 31–40 %; pale pink, 41–50 %; pale cyan, 50–60 %; bright blue, 61–80 %; dark blue, 81–100 %. Note: Y63 was also mutated to Phe, which caused a reduction to 76 % of that of the wild type. b Sequence conservation. Same as in panel A, except that the structure is colored by increasing level of conservation, ramped from teal (low conservation) over cyan, white, and pink to purple (high conservation). c Effect of mutations on activity and binding. kcat/KM values from the suc-ALPF-pNA tetrapeptide assay plotted against FK506-binding protein (FKBP) domain-specific KD values of the S2 peptide. Full-length TtSlyD (TtSlyDFL) is shown in black (labeled wt), TtSlyD constructs with the insert-in-flap (IF) domain replaced by the flap loop from human FKBP12 (TtSlyDΔIF) are shown in gray (labeled ΔIF), IF domain mutants are shown in red, FKBP-domain mutants in blue, and linker mutants in green. The solid gray curve crossing the TtSlyDFL wild-type data point shows the result of varying KM (taken to be equal to KD) only. The dashed gray curve was generated with kcat = 0.5 · kcat (wild type). The effect of all mutations close to the solid curve (Y13F, N35A, and A78G) can be explained primarily by binding, assuming that the effects on KD and KM are the same. Mutations with data points falling on the dashed curve (Y63F, H119A, D23A, I37G, M96A, and Y92A) can be seen to reduce kcat by a factor of two compared to the wild type, while showing variable binding strength. F128A and Y63A have apparently greater effects on kcat, highlighting the importance of these residues