Comparative Binding Energy (COMBINE) Analysis of Barnase-Barstar Interfacial Mutants

1. Comparative Binding Energy (COMBINE) Analysis of Barnase-Barstar Interfacial Mutants Binding features: • High binding affinity (Kd=10-14 M) • Polar binding interface: positive(+2e) on BN, negative (-6e) on BS • Hot-spots Residues at the interface barstar barnase

2. 65 complexes with interfacial mutations Barnase Barstar Arg59 Trp44 Glu80 Thr42 His102 Glu73 Asp39 Glu76 Tyr29 Arg87 Trp38 Lys27 Arg83 Asp35

3. 65 complexes with different interfacial mutations

4. 65 complexes with different interfacial mutations

5. Electrostatic Desolvation Free Energy Calculation • UHBD6.1: Solving the Poisson-Boltzmann equation using a finite difference method

6. Electrostatic Eesolvation Energy of Barnase R2<0.1 Electrostatic Eesolvation Energy of Barstar R2=0.41

7. Electrostatic Interaction Energy between Barnase and Barstar R2=0.63 Electrostatic Binding Free Energy R2=0.35

8. Energy Decomposition to each BN-BS residue pair • Each complex was energy-minimized in AMBER7.0 with AMBER94 ff • Lennard-Jones interactions were calculated for each BN-BS residue pair (110 x 89 = 9790) • Coulomic interactions were calculated for each BN-BS residue pair • (110 x 89 = 9790) • Desolvation energies of BN and BS were calculated in UHBD6.1 Each complex: 19582 (=9790+9790+2) energy terms

9. Chemometric Analysis • Principal Component Analysis (PCA) • Partial Least Squares Analysis (PLS) Contribution of translational and rotational entropy change Contribution of each energy term: Importance of each residue pair

10. Table 1. Predictive Performance of the COMBINE Model

11. Normalized Coefficients at 6 LV Barstar Barnase