Role of electrostatics in differential binding of RalGDS to Rap mutations E30D and K31E investigated by vibrational spectroscopy of thiocyanate probes.

The human protein Rap1A (Rap) is a member of the Ras superfamily of GTPases that binds to the downstream effector Ral guanine nucleotide dissociation stimulator (RalGDS). Although Ras and Rap have nearly identical amino acid sequences and structures along the effector binding surface, the charge reversal mutation Rap K31E has previously been shown to increase the dissociation constant of Rap-RalGDS docking to values similar to that of Ras-RalGDS docking. This indicates that the difference in charge at position 31 could provide a mechanism for Ral to distinguish two structurally similar but functionally distinct GTPases, which would be of vital importance for appropriate biological function. In this report, vibrational Stark effect spectroscopy, dissociation constant measurements, and molecular dynamics simulations were used to investigate the role that electrostatic field differences caused by the charge reversal mutation Rap K31E play in determining the binding specificity of RalGDS to Rap versus Ras. To do this, six variants of RalGDS carrying a thiocyanate electrostatic probe were docked with three Rap mutants, E30D, K31E, and E30D/K31E. The change in absorption energy of the thiocyanate probe caused by RalGDS docking to these Rap variants was then compared to that observed with wild-type Ras. Three trends emerged: the expected reversion behavior, an additive behavior of the two single mutations, and cancelation of the effects of each single mutation in the double mutant. These observations are explained with a physical model of the position of the thiocyanate probe with respect to the mutated residue based on molecular dynamics simulations.

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