Improve the interface between aprotinin and Dengue protease in structure PDB:3U1J.
This interface is partially fortuitous as aprotinin, also known as bovine pancreatic trypsin inhibitor, is a broad spectrum serine protease inhibitor. Therefore the is likely room for improvement. The reason why this was requested was told to me but I forgot. That the active site is occluded is not a problem I am told.
- Minimize
- Point mutation scan
- FastDesign without cysteines
- FastDesign interface
Standard, coordinate restrained
Code: 01-minimise.py
The protein has 3 disulfide bonds (C5-C55, C30-C51, C14-C38). There are a few designs were cysteine to alanine variants are made, such as PDB:2ZJX. Cysteine to alanine is conservative, but might not be the best.
Code: 02-remove_cystine.py
Rosetta Relax with design found these pairs were good:
- C5V C55A
- C14A C38V
- C30A C51T
Together the predicted abs ∆G goes from -745 to -764 kcal/mol.
Code: 03-scan.py
NB. All designs were on the relaxed wild type, not a C5V C14A C30A C38V C51T C55A variant
| mutations | complex_ddG | interface_ddG |
|---|---|---|
| I18L | 1.4 | -2.6 |
| I19W | 1.6 | -2.1 |
| G36G* | -0.5 | -2 |
| P13E | 1.7 | -1.5 |
| R17M | 1.1 | -1.4 |
| R17L | -0.2 | -1.3 |
| R17Q | 0.9 | -0.7 |
| P13N | 1.3 | -0.4 |
| R20A | 1.3 | -0.3 |
| R17A | 1.8 | -0.2 |
| Y21F | 0.3 | -0.1 |
∗ G36G is a noise mutation. This is unfortunate. The controls are as follows:
| mutations | complex_ddG | interface_ddG |
|---|---|---|
| A16A | 3.4 | -2.1 |
| G36G | -0.5 | -2 |
| G37G | 0 | -0 |
| wt | -0 | 0 |
| G12G | 0.5 | 0 |
| K15K | -1 | 0 |
| R17R | 0.6 | 0 |
| K46K | 0.2 | 0 |
| T11T | 0.6 | 0.1 |
| F45F | 0.1 | 0.1 |
| P13P | 0.1 | 0.1 |
| R20R | 0.1 | 0.1 |
| Y21Y | 0.3 | 0.1 |
| I18I | 0.9 | 0.1 |
| I19I | 1 | 0.1 |
| T32T | 0.8 | 0.3 |
| V34V | 2.6 | 0.4 |
This is likely due to the constrained minimisation. Bar for G36G and A16A the noise is low.
Code: 04-fastdesign.py
| mutations | complex_ddG | interface_ddG |
|---|---|---|
| T11E P13W R17L Y21F T32A V34I G36A | 0.2 | -6.6 |
| T11L R17L I18L I19Y R20A T32V V34I G36A | -2.9 | -5.6 |
| T11L R17L I18L I19Y R20A Y21W T32V V34I G36A | -1.9 | -5.4 |
| T11E P13W R17L R20V Y21W T32A V34I G36A | 0.7 | -5 |
| T11V R17L I18L I19F R20A Y21W T32V V34I G36A S47T | -2.5 | -4.8 |
| T11S R17L I19F R20V T32V V34I G36A S47K | -4 | -4.8 |
| T11L R17L I18L I19Y R20A Y21W T32V V34I G36A S47T | -2.9 | -4.6 |
| T11E P13N R17L I19W R20A Y21A T32Y V34I G36A S47K | -4.8 | -4.4 |
| T11E P13W R17L Y21W T32A V34I G36A S47T | 1.5 | -4.3 |
| T11V P13N R17L I18L I19Y R20V Y21W T32V V34I G36A S47T | -2 | -4.3 |
| P13N R17L R20A Y21W T32A V34I | -2.6 | -4.2 |
| T11L R17F I18L I19L R20A T32V V34I G36A S47K | -6.3 | -4 |
| P13V R17L R20A Y21W T32A V34I | -2.8 | -3.7 |
| T11V R17L I18L I19F R20V Y21W T32V V34I G36A S47T | -0.7 | -3.7 |
| T11V R17L I18L I19Y R20V Y21W T32V V34I G36A S47T | -2 | -3.7 |
| T11E R17L I19Y R20A Y21W T32V V34I G36A S47T | -3.3 | -3.6 |
| T11E R17L I19W R20A Y21A T32Y V34I G36A S47K | -2.4 | -3.5 |
| T11V R17L I18L I19F Y21W T32V V34I G36A S47T | -3 | -3.4 |
| R17L R20V Y21F T32A V34I | -1.8 | -3.3 |
| R17L I19W R20A Y21A T32Y V34I | -3.9 | -3.2 |
| T11L R17L I19F R20V Y21W T32V V34I G36A S47T | -3.6 | -3.2 |
| T11L R17Y I18L I19L Y21W T32V V34I G36A S47T | -3.6 | -3.1 |
| T11E P13N R17F I19L R20V Y21W T32V G36A S47T | -3.1 | -3.1 |
| P13N R17L R20A Y21F T32A V34I | -2.2 | -3.1 |
| T11E R17L I19W R20V Y21A T32Y V34I G36A S47K | -4.7 | -3 |
| T11E R17F I19L R20V T32V G36A S47T | -1.8 | -2.9 |
| T11E R17Y I19Y R20A Y21A T32Y V34I G36A S47H | -3.1 | -2.9 |
| Y21A Y23W G36A R39D A48L T54V G56A | -10.7 | -2.9 |
| T11V R17L I19W R20V Y21A T32Y V34I G36A S47K | -3.7 | -2.8 |
| R17Y R20V Y21F T32A | -1 | -2.8 |
| T11V R17F I18L I19L R20A Y21W T32V G36A | -2 | -2.8 |
| I18L | 1.4 | -2.6 |
| R17L I19W R20V Y21A T32Y V34I S47K | -4.9 | -2.5 |
| R17L I19W R20V Y21W T32V V34I S47T | -1.8 | -2.5 |
| Y21A Y23F G36A R39D A48L T54V G56S | -11.9 | -2.5 |
| T11S R17L Y21W T32A V34I G36A | -1.7 | -2.4 |
| T11V R17L I19L R20V Y21W T32V V34I G36A S47T | -2.5 | -2.4 |
| T11V R17L I19L R20V Y21W T32V V34I G36A | -1.9 | -2.4 |
| T11E R17F R20V Y21W T32A V34I G36A | -0.1 | -2.3 |
| P13V R17L R20A Y21F T32A V34I | -2.2 | -2.2 |
| P13V R17Y R20A Y21W T32A | -1.5 | -2.2 |
| R17Y I18L Y21F T32A | -1.3 | -2.2 |
| T11E P13N R17F I18L R20Y Y21W T32P V34I G36A | -5.1 | -2.2 |
| T11L R17Y I18L I19V R20A G36A | -2.7 | -2.2 |
| T11E R17F R20A Y21W T32A V34I G36A | -1.7 | -2.1 |
| T11L R17L I18L Y21W T32A V34I G36A | -0.4 | -2.1 |
| T11E R17L R20A Y21F T32A V34I G36A | -0.7 | -2.1 |
| T11E K15R R17L I19W R20V Y21A T32Y V34I G36A S47K | -2.7 | -2.1 |
| I19W | 1.6 | -2.1 |
| P13V R17F R20A Y21W T32A V34I | -1.7 | -2.1 |
| T11E R17L R20V Y21F T32A V34I G36A | -0 | -2 |
| -0.5 | -2 | |
| T11E R17F R20V Y21W T32A V34I G36A S47T | 1 | -2 |
| R17L R20A Y21F T32A V34I | -2 | -2 |
| T11E R17L I18L R20V Y21A T32Y V34I G36A S47T | -0.7 | -2 |
| R17Y Y21F T32A | -2.2 | -2 |
| T11E P13N R17F R20V Y21W T32A V34I G36A S47T | 0.6 | -1.9 |
| P13V R17L R20V Y21F T32A V34I | -1.7 | -1.9 |
| P13N R17L I18L Y21W T32A V34I | -1.2 | -1.9 |
| R17L I18L Y21F T32A V34I | -0.8 | -1.8 |
| T11V R17Y I18L I19L Y21W T32V V34I G36A | -3.3 | -1.8 |
| R17F R20V Y21W T32A V34I S47T | -1.6 | -1.8 |
| T11L R17L R20A Y21W T32A V34I G36A S47T | -1 | -1.8 |
| T11V R17F I19L R20V Y21W T32V G36A S47T | -3 | -1.8 |
| T11V R17F R20V Y21W T32A V34I G36A | 1 | -1.8 |
| T11V R17F I18L I19L Y21W T32V V34I G36A S47T | -3.2 | -1.8 |
| T11V R17Y I18L I19L R20V Y21W T32V G36A S47T | -1.3 | -1.8 |
| P13N R17L R20A Y21A T32M V34I | 1.4 | -1.8 |
| T11V R17F I18L I19L Y21W T32V G36A S47T | -2.6 | -1.7 |
| T11S R17F R20V Y21W T32A V34I G36A | -0.6 | -1.7 |
| P13N R17L R20V Y21W T32A V34I | -1.9 | -1.7 |
| T11L R17L R20A T32A V34I G36A | -1.5 | -1.7 |
| T11S R17F R20A Y21W T32A V34I G36A | -2 | -1.7 |
| P13D R17Y R20A Y21W T32A | -2.3 | -1.7 |
| T11E P13D R17L R20A Y21F T32A V34I G36A | -0.7 | -1.7 |
| R17L I18L R20A T32A V34I | -1.6 | -1.7 |
| T11E R17Y Y21F T32A G36A | -0.5 | -1.7 |
| R17L R20A T32A V34I | -0.9 | -1.7 |
| R17F I18L Y21W T32A V34I | -2.1 | -1.6 |
| R17F I18L I19L Y21W T32V V34I G36A | -2.9 | -1.6 |
| R17F I18L Y21W T32A V34I S47T | -1.6 | -1.6 |
| P13D R17L I18L Y21F T32A V34I | -2 | -1.6 |
| T11S R17F Y21W T32A V34I G36A | -1.2 | -1.6 |
| T11V R17L R20V Y21W T32A V34I G36A S47T | -0.5 | -1.6 |
| R17L I18L Y21F T32A V34I S47T | -1.3 | -1.5 |
| T11S R17L I19W R20V Y21A T32Y V34I G36A S47T | -4.3 | -1.5 |
| R17L R20A Y21W T32A V34I | -2.6 | -1.5 |
| T11E R17L I18L Y21F T32A V34I G36A | 0.2 | -1.5 |
| P13E | 1.7 | -1.5 |
| R17L I18L T32A V34I S47T | -1.2 | -1.5 |
| T11E P13V R17F I19L R20V Y21W T32V G36S S47T | -1.7 | -1.5 |
| T11E R17F I18L T32A V34I G36A | -1.3 | -1.5 |
| R17L I18L T32A V34I | -1.5 | -1.4 |
| T11V R17L I18L Y21W T32A V34I G36A S47T | -1 | -1.4 |
| R17L Y21F T32A V34I | -2.4 | -1.4 |
| R17L I18L Y21W T32A V34I G36A | -1.4 | -1.4 |
| R17M | 1.1 | -1.4 |
| R17L I19W R20V Y21W T32A V34I S47T | -2.3 | -1.4 |
| T11E R17Y I18L Y21W T32A V34I G36A S47T | -1.1 | -1.3 |
| T11V A16V R17L I18L R20V Y21A T32Y V34I G36A S47K | -1.8 | -1.3 |
For analysis of repetitions and epistasis, see email.
Possible improvements:
- I did not run an MD run or Rosetta Backrub to get alternative conformations of the protein for better sampling.
- I forgot to calculate the monomer ∆G...
- I can re-run everything on the variant without cysteines...