Determining the Electrostatic Contributions of GTPase-GEF Complexes on Interfacial Drug Binding Specificity: A Case Study of a Protein–Drug–Protein Complex

Understanding the factors that contribute to specificity of protein–protein interactions allows for design of orthosteric small molecules. Within this environment, a small molecule requires both structural and electrostatic complementarity. While the structural contribution to protein–drug–protein specificity is well characterized, electrostatic contributions require more study. To this end, we used a series of protein complexes involving Arf1 bound to guanine nucleotide exchange factors (GEFs) that are sensitive or resistant to the small molecule brefeldin A (BFA). By comparing BFA-sensitive Arf1-Gea1p and Arf1-ARNO with different combinations of four BFA sensitizing ARNO mutations (ARNOwt, ARNO1M, ARNO3M, and ARNO4M), we describe how electrostatic environments at each interface guide BFA binding specificity. We labeled Arf1 with cyanocysteine at several interfacial sites and measured by nitrile adsorption frequencies to map changes in electric field at each interface using the linear Stark equation. Temperature dependence of nitrile vibrational spectra was used to investigate differences in hydrogen bonding environments. These comparisons showed that interfacial electric field at the surface of Arf1 varied substantially depending on the GEF. The greatest differences were seen between Arf1-ARNOwt and Arf1-ARNO4M, suggesting a greater change in electric field is required for BFA binding to Arf1-ARNO. Additionally, rigidity of the interface of the Arf1-ARNO complex correlated strongly with BFA sensitivity, indicating that flexible interfaces are sensitive to disruption upon orthosteric small molecule binding. These findings demonstrate a qualitatively consistent electrostatic environment for Arf1 binding and more subtle differences preventing BFA specificity. We discuss how these results will guide improved design of other small molecules that can target protein–protein interfaces.