Fig. 4: The N-terminal residue D26 interacts with residues R89, K54, R55, R61, and R62 on the concave surface of Spy.

a Surface representations of Spy_29-124 (PDB: 3O39) showing the side chains of all the positively charged residues in the stick model. The side chain nitrogen atoms of these residues are colored blue. Residues selected for further testing are labeled in red. b Correlations between the intramolecular PRE intensity ratios of different Spy_{1-124 CI} variants and the PRE signals of Spy_{1-124 CI}. The RMSD values of the PRE intensity ratios are presented for each comparison group. Gray dashed lines represent the deviation from the diagonal (black dashed line) for a value of 0.78, which is the RMSD value between Spy_{1-124 CI} and the control protein Spy_{1-124 CI K75D}. For the plot of Spy_{1-124 CI} and Spy_{1-124 CI K75D}, most of the data points are distributed between the two gray lines. Thus, these lines help to make a visual evaluation for the distribution of data points. c Comparisons of dissociation rate constants k_off of Spy variants to the k_off of Spy wild type (mean ± SD, n = 3 independent experiments for WT, R61D R62D, and R89D, n = 4 independent experiments for K54D R55D, n = 2 independent experiments for the other variants, individual data points are shown; unpaired two-tailed Student’s t test). Statistical analysis was performed with Graphpad Prism 9.1.0. d Position of the MTSL spin label on the concave surface of Spy during MD simulations of Spy_CI and its variants. The two monomers of Spy are shown in white and gray, respectively. A total of 1000 frames are uniformly resampled in each 1-μs trajectory, and the nitroxide oxygen (O1) atoms from each monomer are represented by cyan and orange spheres, respectively. Source data are provided as a Source Data file.