Promiscuous Protein Binding as a Function of Protein Stability Ruth Cohen-Khait, Orly Dym, Shelly Hamer-Rogotner, Gideon Schreiber  Structure  Volume 25, Issue 12, Pages 1867-1874.e3 (December 2017) DOI: 10.1016/j.str.2017.11.002 Copyright © 2017 Elsevier Ltd Terms and Conditions

Structure 2017 25, 1867-1874.e3DOI: (10.1016/j.str.2017.11.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 The Selected Mutations Visualized on the Structure of TEM1 The identities of the mutations present in the four selected clones are provided in the table. The position of each of the selected mutations is represented as orange sticks on the TEM1-WT scaffold (PDB: 1JTG chain A). Structure 2017 25, 1867-1874.e3DOI: (10.1016/j.str.2017.11.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Crystal Structure of the T1 G268A (Green) Binding TEM1-WT (Wheat) (A) The two similar proteins interact through a pseudo-2-fold symmetry, which results in formation of β-sheet main-chain H bonds between the proteins (lower inset: blue, nitrogen; red, oxygen; magenta dashed lines, main-chain H bonds). In addition, the interaction is supported by an interaction formed between the N′ helix of TEM1-WT and the C′ helix of T1 G268A. The upper inset shows the two salt bridges stabilizing the N′ helix in TEM1-WT (PDB: 1BTL); E37 interacts with R61 and E58 forms a salt bridge with the NH2 terminus. (B) Interacting residues from both proteins as predicted by AquaProt (Reichmann et al., 2007) are presented in red (TEM1-WT) and light blue (T1 G268A). The chemical bond formed between the interacting residues is indicated by the type of their connecting line: black, hydrophobic; blue, polar. Lines denote interaction via side chain and arrows denote interaction via main chain. Structure 2017 25, 1867-1874.e3DOI: (10.1016/j.str.2017.11.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Superposition of TEM1-WT (Magenta) on T1 G268A (Light Blue) The structure of T1 G268A is in complex with TEM1-WT (green), indicating the steric clash between the two N′ helices. Structure 2017 25, 1867-1874.e3DOI: (10.1016/j.str.2017.11.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 TEM1-TEM1 Binding Specificity FRET 530/480 ratio was measured for TEM1-TEM1 binding of the given clones. For each pair one protein was fussed to YPET (acceptor emission at 530 nm) and the second protein was attached to CyPET (donor emission at 480 nm). The concentration of both proteins was 1 μM. The fluorescence of separate CyPET and YPET fused TEM1 proteins were subtracted from all data points. The dashed line represents the highest FRET value of the non-specific binding. Figure S4 provides measurements done at concentrations between 0.5 and 8 μM (without subtraction of separate CyPET and YPET), showing an average difference of 1.8 (±SE 0.4) between specific and non-specific binding. Structure 2017 25, 1867-1874.e3DOI: (10.1016/j.str.2017.11.002) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 TEM1 In Vivo Activity Assay E. coli Bl21 transformed with plasmids containing TEM1 mutants were grown on LB-agar plates with different ampicillin concentrations (0, 20, 100, and 500 μg/mL). Transformation of a plasmid not containing TEM1 served as negative control. TEM1-HS and T1 contain stabilizing mutations while WT does not. T-1 and WT clones were mutated as indicated. Structure 2017 25, 1867-1874.e3DOI: (10.1016/j.str.2017.11.002) Copyright © 2017 Elsevier Ltd Terms and Conditions