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

barstar

barnase

• High binding affinity (Kd=10-14 M)

•Polar binding interface: positive(+2e) on BN, negative (-6e) on BS

•Hot-spots Residues at the interface

Binding features:

65 complexes with interfacial mutations

Arg59

Glu73

Arg83

His102

Arg87Lys27

Glu80

Glu76Trp38

Thr42

Tyr29Asp39

Asp35

Trp44

Barnase

Barstar

65 complexes with different interfacial mutationsNo. Complex Exp. Binding delta_G Desol_BN Desol_BS Elec_inter Elect_binding_G1 wtwt -19 83.29 75.7 -149.629 9.3592 wtK27Awm -13.6 61.78 64.66 -121.787 4.6583 wtR59A -13.8 67.68 69.38 -120.451 16.6084 D39AR59A -7.7 60.52 23.06 -45.371 38.2045 wtR87A -13.5 75.58 70.4 -111.981 33.9996 wtH102Awm -12.9 75.93 73.32 -131.979 17.2797 Y29FWTWMx -19.1 74.54 66.27 -129.535 11.2748 Y29AWT -15.6 76.86 66.26 -131.241 11.889 D35Awtwm -14.5 82.35 51.48 -121.022 12.81410 W38FWT -17.4 80.2 72.81 -147.522 5.4911 D39Awt -11.3 78.3 44.99 -89.331 33.9612 T42Awt -17.2 82.07 73.01 -142.04 13.04213 W44FWT -19 82.54 74.96 -148.134 9.3714 E76wt -17.7 76.34 70.45 -133.19 13.59515 E80AWT -18.5 77.67 70.21 -140.513 7.36616 Y29AK27AWM -10.4 56.34 56.68 -104.906 8.11417 D35AK27Awm -9.5 58.45 41.3 -91.552 8.1918 W38FK27AWM -12.6 59.29 62.5 -115.126 6.67119 D39AK27Awm -10.8 52.85 41.48 -74.609 19.72320 T42AK27Awm -13.3 59.23 63.75 -118.2 4.77321 E76AK27AWM -12.3 54.36 59.53 -103.754 10.13722 E80AK27Awm -13.5 58.31 61.2 -116.631 2.87723 Y29AR59A -10.9 61.75 60.39 -102.207 19.92624 D35AR59AWM -12.7 63.45 48.5 -100.483 11.45825 W38FR59A -12.8 66.82 68.36 -119.921 15.25526 T42AR59A -12.2 65.87 66.64 -112.196 20.30827 E76AR59A -14.1 65.73 66.76 -118.262 14.2328 E80AR59A -13.9 65.04 67.33 -118.106 14.25729 Y29AR83Q -10.7 65.36 62.63 -85.188 42.79730 D35AR83QWM -9.4 67.83 47.64 -72.423 43.046

32 D39AR83Q -12.6 55.34 42.26 -75.182 22.41633 T42AR83Q1wm -12.9 66.04 68.45 -90.987 43.50634 E76AR83Q -12.3 63.86 67.18 -83.349 47.68635 E80AR83Q -13.3 68.43 69.37 -96.995 40.80236 Y29AR87A -11 70.18 61.85 -102.068 29.96337 T42AR87A -12 72.96 68.27 -103.662 37.56838 E76AR87A -12.2 69.28 66.45 -96.035 39.69639 E80AR87A -12.9 72.71 68.65 -107.539 33.8240 Y29AH102AWM -12.7 69.04 62.5 -112.803 18.73641 Y29FH102AWMx -13.5 69.12 63.08 -112.538 19.66942 W38FH102AWM -11.4 75.04 71.71 -130.846 15.90843 D39AH102Awm -10.1 70.31 43.2 -80.129 33.37944 T42AH102Awm -10.9 74.47 70.98 -123.278 22.17145 E76AH102AWM -11.5 71.25 68.4 -117.52 22.12846 E80AH102AWM -12.3 74.11 70.37 -128.723 15.75747 D35AE73Wwm -13.3 97.49 51.49 -141.319 7.66148 D39AE73A -11.9 86.44 44.05 -95.671 34.82549 D39AE73Q -11.8 98.07 44.89 -99.043 43.91850 E76AE73Q -15.5 100.37 71.19 -159.859 11.70451 wtE73A -16.7 91.86 70.78 -166.59 -3.95352 wtE73C -16.5 97.02 73.44 -171.877 -1.4253 wtE73F -16.8 104.48 76.16 -181.322 -0.67454 wtE73Q -17.6 104.47 75.68 -174.595 5.55355 wtE73S -16 93.75 71.2 -168.858 -3.91356 wtE73Y -16.6 96.73 73.9 -176.256 -5.62357 D35AE73Awm -12.6 95.94 48.24 -133.672 10.50458 D35AE73Fwm -12.6 103.76 52.4 -142.665 13.49159 D39AE73F -11.6 95.04 44.16 -96.927 42.27660 D39AR87A -11.9 68.25 44.93 -84.455 28.72461 E76AE73W -15.5 94.18 71.18 -163.989 1.3762 W38FR87A -12 75.12 69.55 -110.615 34.04963 WTE73W -17.4 97.84 75.42 -179.2 -5.93464 WTR83Q -13.6 67.07 70.61 -95.287 42.39765 D39AE73W -12.4 91.53 44.32 -97.266 38.585

65 complexes with different interfacial mutations

Electrostatic Desolvation Free Energy Calculation

• UHBD6.1: Solving the Poisson-Boltzmann equation using a finite difference method

PdesoleleG

LPele

Ldesolele

Pdesolele

bindele EGGG

LdesoleleG

LPeleE

LPeleE

Electrostatic Eesolvation Energy of Barnase

Electrostatic Eesolvation Energy of Barstar

R2<0.1

R2=0.41

R2=0.63

R2=0.35

Electrostatic Interaction Energy between Barnase and Barstar

Electrostatic Binding Free Energy

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

Chemometric Analysis

• Principal Component Analysis (PCA)

• Partial Least Squares Analysis (PLS)

CuwuwGwGwGi

elei

elei

i

vdwi

vdwi

Ldesol

Ldesol

Rdesol

Rdesol

Contribution of each energy term: Importance of each residue pair

Contribution of translational and rotational entropy change

Data set

#Complexes #LV R2 Q2 SDEP

(kcal/mol)

Constant C

(kcal/mol)

G 65

4 0.65 0.57 1.67 -2.928

5 0.85 0.74 1.29 +1.63

6 0.91 0.86 0.97 +5.364

7 0.93 0.88 0.87 +6.351

Table 1. Predictive Performance of the COMBINE Model

D39

D35

G31

T42

A36

W38

E76

E80

E76vdw

Y29vdw

D39vdw

T42vdwR83 R87 R59 H102 D75 K27 E73

R59vdw

H102vdw

R83vdw

K27vdw

-0.5

0

0.5

1

1.5

2

Normalized Coefficients at 6 LV

Barstar Barnase