Affinity Increase.Protocol Documentation
The Increase Affinity protocol is based on the premise that increasing buried hydrophobic surface area and/or decreasing
buried hydrophilic surface area will generally lead to enhanced binding affinity provided steric clashes are not introduced
and buried polar groups are not left without a hydrogen bond partner. All point mutations at the protein-protein interface
that fit the above criteria are tried, and the resulting changes in protein-protein binding free energies (ddGbinding) are
computed. ddGbinding is computed using the following equation:
ddGbinding=dGmut-dGwt
where dGmut/dGwt are the binding energies computed for the mutant and wild type structures respectively.
An energies table is output by Rosetta. The results in the table are sorted, with the point mutations that are predicted to yield
the greatest increase in binding affinity at the top of the table. The results are also filtered such that point mutations that
are predicted to give a ddGbinding of greater than -0.5 kcal/mol are not included.
Tendencies of the Increase Affinity Protocol:
This protocol assumes that the conformation of the unbound state is the same as the bound state,
thus losing information about the stability of individual partners in the ddGbinding predictions.
We therefore include ddGpartner A and ddGpartner B in the output table. We have also found that the
energetic penalty for removing a hydrogen bond in an unminimized crystal structure can be insignificant
compared to the other energy terms, resulting in a false prediction that the mutation is stabilizing
to the complex. In addition, if a polar residue has a large number of neighbors (~>20), that residue
can be considered very buried, and is likely participating in a hydrogen bond and should probably not be
mutated. We include the ddGh-bond and number of neighbors to help the user screen for these types of
destabilizing mutations.
The following additional filters may be applied at the user's discretion, to address the above mentioned
issues in an automated way: 1) require the ddGh-bond, the hydrogen bond energy term, to be zero or less,
directing the results to exclude mutations that remove a side-chain involved in a hydrogen bond, and
2) require that the ddGpartner A, and ddGpartner B be less than or equal to 1.0 in an effort to remove mutations
that are predicted to significantly destabilize one of the individual chains.
Our work indicates that adding these filters does tend to increase the rate of success, but at the expense
of the number of predictions. Some protein-protein interfaces yield so few predictions that pass these filters
that it becomes necessary to leave one or both of them off.
INTOUT file from Increase Affinity Protocol:
Mutation ddG_bind ddG_partnerA ddG_partnerB #Neighbors ddG_h_bond
98B N>L: -9.7 0.0 0.3 14 0.0
98B N>Y: -9.7 0.0 0.3 14 0.0
98B N>F: -9.6 0.0 0.4 14 0.0
98B N>M: -9.4 0.0 0.6 14 0.0
The mutation data is in following format:
98B N>L where the chain is B, the wild type amino acid is N, the sequence position is 98,
and the mutant amino acid is L.
Designing affinity enhancing mutations with multi-chain PDBs
To design stability enhancing mutations on PDB files with 3 or more chains, you must reconstruct the PDB file with
a TER between the two "sides" of the interface. For example, take a PDB file with 4 chains: A, B, C, and D.
Assume you want to design stability enhancing mutations between the interface formed by chains A and C together with
chains B and D together. You will need to edit your PDB file so that all the ATOM lines from chains A and C are
together, followed by a line containing only TER, followed by all the ATOM lines in chains B and D.
Below is an truncated example of how the PDB should look to design between chains AC and BD. Note that the atom and
residue numbering in the PDB file do not have to be in order.
ATOM 1 N SER A 11 84.265 65.648 63.661 1.00 64.25 N
ATOM 2 CA SER A 11 83.271 66.744 63.480 1.00 63.79 C
ATOM 3 C SER A 11 83.851 67.907 62.673 1.00 63.01 C
ATOM 4 O SER A 11 83.138 68.855 62.329 1.00 63.82 O
ATOM 7 N LEU A 12 85.145 67.835 62.374 1.00 60.18 N
ATOM 8 CA LEU A 12 85.801 68.876 61.590 1.00 57.24 C
ATOM 9 C LEU A 12 85.558 68.630 60.108 1.00 53.24 C
ATOM 10 O LEU A 12 85.292 67.499 59.697 1.00 53.48 O
ATOM 3442 N LYS C 94 94.253 40.203 50.905 1.00 66.18 N
ATOM 3443 CA LYS C 94 94.422 38.755 50.902 1.00 70.49 C
ATOM 3444 C LYS C 94 94.934 38.311 49.529 1.00 72.66 C
ATOM 3445 O LYS C 94 94.429 38.754 48.498 1.00 72.25 O
ATOM 3451 N PRO C 95 95.957 37.440 49.504 1.00 74.91 N
ATOM 3452 CA PRO C 95 96.555 36.928 48.265 1.00 76.39 C
ATOM 3453 C PRO C 95 95.554 36.281 47.309 1.00 78.07 C
ATOM 3454 O PRO C 95 95.707 35.073 47.022 1.00 79.37 O
TER
ATOM 1391 N SER B 11 105.103 72.699 78.897 1.00 51.29 N
ATOM 1392 CA SER B 11 106.135 71.642 79.073 1.00 49.75 C
ATOM 1393 C SER B 11 106.878 71.399 77.761 1.00 47.71 C
ATOM 1394 O SER B 11 107.895 70.707 77.733 1.00 47.98 O
ATOM 1397 N LEU B 12 106.365 71.963 76.672 1.00 44.31 N
ATOM 1398 CA LEU B 12 107.001 71.792 75.371 1.00 41.63 C
ATOM 1399 C LEU B 12 108.215 72.701 75.245 1.00 39.97 C
ATOM 1400 O LEU B 12 108.289 73.748 75.896 1.00 39.19 O
ATOM 3459 N SER D 4 80.060 73.057 67.538 1.00 61.21 N
ATOM 3460 CA SER D 4 80.054 73.502 66.114 1.00 60.96 C
ATOM 3461 C SER D 4 81.475 73.642 65.569 1.00 61.03 C
ATOM 3462 O SER D 4 81.744 73.267 64.429 1.00 61.61 O
ATOM 3465 N ARG D 5 82.374 74.182 66.391 1.00 60.41 N
ATOM 3466 CA ARG D 5 83.773 74.377 66.011 1.00 60.25 C
ATOM 3467 C ARG D 5 84.673 74.232 67.247 1.00 60.14 C
ATOM 3468 O ARG D 5 84.180 74.101 68.370 1.00 59.86 O