Abstract
The dissociation constant for an ionizable ligand binding to a receptor is dependent on its charge and therefore on its environmentally-influenced pKa value. The pKa values of sphingosine 1-phosphate (S1P) were studied computationally in the context of the wild type S1P1 receptor and the following mutants: E3.29Q, E3.29A, and K5.38A. Calculated pKa values indicate that S1P binds to S1P1 and its site mutants with a total charge of -1, including a +1 charge on the ammonium group and a -2 charge on the phosphate group. The dissociation constant of S1P binding to these receptors was studied as well. The models of wild type and mutant proteins originated from an active receptor model that was developed previously. We used ab initio RHF/6-31+G(d) to optimize our models in aqueous solution, where the solvation energy derivatives are represented by conductor-like polarizable continuum model (C-PCM) and integral equation formalism polarizable continuum model (IEF-PCM). Calculation of the dissociation constant for each mutant was determined by reference to the experimental dissociation constant of the wild type receptor. The computed dissociation constants of the E3.29Q and E3.29A mutants are three to five orders of magnitude higher than those for the wild type receptor and K5.38A mutant, indicating vital contacts between the S1P phosphate group and the carboxylate group of E3.29. Computational dissociation constants for K5.38A, E3.29A, and E3.29Q mutants were compared with experimentally determined binding and activation data. No measurable binding of S1P to the E3.29A and E3.29Q mutants was observed, supporting the critical contacts observed computationally. These results validate the quantitative accuracy of the model.