Role of Electrostatics in Differential Binding of RalGDS to Rap Mutations E30D and K31E Investigated by Vibrational Spectroscopy of Thiocyanate Probes

Abstract

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.

Cyanylated Cysteine Reports Site-Specific Changes at Protein–Protein-Binding Interfaces Without Perturbation

“…Guideline 3 above is drawn partly from the Webb group’s results, where large electrostatic changes due to the probe group at a few sites disrupted the functional cooperation between Ras and its effectors. 31 − 33 The R16C* mutation in the CaM/M13 system here was not fatal in line with other previous work, 61 but such electrostatic perturbations can also have large effects on the conformational ensembles of disordered domains in particular 77 (a class of domains that includes many CaM-binding sequences) and should generally be avoided.…”

Site-Specific Infrared Probes of Proteins

“…Next, we describe examples showing how the C≡N stretching vibration can be used to interrogate the local electric field environment of proteins and, in particular, the role of electrostatics in determining the catalytic activity of enzymes (83–85). In one study, Herschlag and coworkers (86) used Vibrational Stark Effect (VSE) spectroscopy with the SCN probe, in conjunction with NMR measurements, to characterize how modulating the charge of a bound ligand (i.e., phenol) affects the local electrostatic environment in the active site of the bacterial enzyme ketosteroid isomerase.…”

Ester Carbonyl Vibration as a Sensitive Probe of Protein Local Electric Field

“…[1] However, quantifying the local electric field or how it changes inside a protein, especially in a site-specific manner and/or as a function of time, still remains a challenging task. One promising method in this regard is vibrational Stark spectroscopy, [2] which capitalizes on the fact that vibrational transitions have an intrinsic dependence on local electrostatic environment and uses an infrared (IR) probe that has a well-defined, localized vibrational mode to sense local electric field amplitude through the response of the frequency.…”