TY - JOUR
T1 - Structure-based Protocol for Identifying Mutations that Enhance Protein-Protein Binding Affinities
AU - Sammond, Deanne W.
AU - Eletr, Ziad M.
AU - Purbeck, Carrie
AU - Kimple, Randall J.
AU - Siderovski, David P.
AU - Kuhlman, Brian
N1 - Funding Information:
We thank F.S. Willard and C.R. McCudden for aid in protein expression and purification. We thank the Renaissance Computing Institute for providing access to their computational resources. This research was supported by an award from the W.M. Keck foundation and the grants GM074268 and GM073960 from the National Institutes of Health.
PY - 2007/8/31
Y1 - 2007/8/31
N2 - The ability to manipulate protein binding affinities is important for the development of proteins as biosensors, industrial reagents, and therapeutics. We have developed a structure-based method to rationally predict single mutations at protein-protein interfaces that enhance binding affinities. The protocol is based on the premise that increasing buried hydrophobic surface area and/or reducing buried hydrophilic surface area will generally lead to enhanced affinity if large steric clashes are not introduced and buried polar groups are not left without a hydrogen bond partner. The procedure selects affinity enhancing point mutations at the protein-protein interface using three criteria: (1) the mutation must be from a polar amino acid to a non-polar amino acid or from a non-polar amino acid to a larger non-polar amino acid, (2) the free energy of binding as calculated with the Rosetta protein modeling program should be more favorable than the free energy of binding calculated for the wild-type complex and (3) the mutation should not be predicted to significantly destabilize the monomers. The performance of the computational protocol was experimentally tested on two separate protein complexes; Gαi1 from the heterotrimeric G-protein system bound to the RGS14 GoLoco motif, and the E2, UbcH7, bound to the E3, E6AP from the ubiquitin pathway. Twelve single-site mutations that were predicted to be stabilizing were synthesized and characterized in the laboratory. Nine of the 12 mutations successfully increased binding affinity with five of these increasing binding by over 1.0 kcal/mol. To further assess our approach we searched the literature for point mutations that pass our criteria and have experimentally determined binding affinities. Of the eight mutations identified, five were accurately predicted to increase binding affinity, further validating the method as a useful tool to increase protein-protein binding affinities.
AB - The ability to manipulate protein binding affinities is important for the development of proteins as biosensors, industrial reagents, and therapeutics. We have developed a structure-based method to rationally predict single mutations at protein-protein interfaces that enhance binding affinities. The protocol is based on the premise that increasing buried hydrophobic surface area and/or reducing buried hydrophilic surface area will generally lead to enhanced affinity if large steric clashes are not introduced and buried polar groups are not left without a hydrogen bond partner. The procedure selects affinity enhancing point mutations at the protein-protein interface using three criteria: (1) the mutation must be from a polar amino acid to a non-polar amino acid or from a non-polar amino acid to a larger non-polar amino acid, (2) the free energy of binding as calculated with the Rosetta protein modeling program should be more favorable than the free energy of binding calculated for the wild-type complex and (3) the mutation should not be predicted to significantly destabilize the monomers. The performance of the computational protocol was experimentally tested on two separate protein complexes; Gαi1 from the heterotrimeric G-protein system bound to the RGS14 GoLoco motif, and the E2, UbcH7, bound to the E3, E6AP from the ubiquitin pathway. Twelve single-site mutations that were predicted to be stabilizing were synthesized and characterized in the laboratory. Nine of the 12 mutations successfully increased binding affinity with five of these increasing binding by over 1.0 kcal/mol. To further assess our approach we searched the literature for point mutations that pass our criteria and have experimentally determined binding affinities. Of the eight mutations identified, five were accurately predicted to increase binding affinity, further validating the method as a useful tool to increase protein-protein binding affinities.
KW - Rosetta molecular modeling software
KW - computational protein design
KW - hydrophobic effect
KW - protein binding hotspots
KW - protein-protein interactions
UR - http://www.scopus.com/inward/record.url?scp=34547571461&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2007.05.096
DO - 10.1016/j.jmb.2007.05.096
M3 - Article
C2 - 17603074
AN - SCOPUS:34547571461
SN - 0022-2836
VL - 371
SP - 1392
EP - 1404
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 5
ER -