Sphingosine 1-phosphate pKa and binding constants: Intramolecular and intermolecular influences

Abstract

The dissociation constant for an ionizable ligand binding to a receptor is dependent on its charge and therefore on its environmentally-influenced pK a value. The pK a values of sphingosine 1-phosphate (S1P) were studied computationally in the context of the wild type S1P 1 receptor and the following mutants: E3.29Q, E3.29A, and K5.38A. Calculated pK a values indicate that S1P binds to S1P 1 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 3-5 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.

Lipid Signaling in Plants

“…LPA and sphingosine-1-phosphate (S1P) are important signaling molecules that bind and activate G-protein-coupled receptors (GPCRs), located in the plasmamembrane and nuclear membrane of cells. This is exactly what recent work by Tigyi and Parrill on the binding of S1P to the S1P 1 receptor showed (Naor et al 2007). Interestingly, the results described above would suggest that LPA and S1P bind to their receptors in a di-anionic form.…”

“…The binding of LPA and S1P to their respective GPCRs critically depends on the phosphomonoester headgroup (Lynch and Macdonald 2002;Sardar et al 2002), and the phosphate-binding region of the LPA receptors all contain two conserved basic residues (one arginine and one lysine residue, (Sardar et al 2002)). Importantly, this work also revealed that the intramolecular hydrogen bond between the hydroxyl group in the backbone of S1P and the phosphomonoester stabilizes the deprotonated form of S1P, exactly what was observed experimentally for the related lipid ceramide-1-phosphate (Cer-1-P; Kooijman et al 2008;Naor et al 2007). This is exactly what recent work by Tigyi and Parrill on the binding of S1P to the S1P 1 receptor showed (Naor et al 2007).…”

“…Recent experimental and computational work by Tigyi and Parrill further substantiate the importance of the phosphomonoester headgroup in lipid-protein interactions and subsequently illustrate the role of intramolecular hydrogen bonds in the dissociation constants of these phosphomonoesters headgroups (Naor et al 2007). LPA and sphingosine-1-phosphate (S1P) are important signaling molecules that bind and activate G-protein-coupled receptors (GPCRs), located in the plasmamembrane and nuclear membrane of cells.…”

Lipid Receptor S1P1 Activation Scheme Concluded from Microsecond All-Atom Molecular Dynamics Simulations

“…They illustrated their findings with a model of the ligand-receptor complex constructed on early rhodopsin model based on distance geometry calculations with hydrogen bonding constraints [51]. Those three residues were also shown as binding S1P in S1P 1 binding site in more recent paper of the same group [52]. The crystal structure of S1P 1 receptor with antagonist ML056 can verify to some extent those findings.…”

Lysophospholipid interactions with protein targets

“…These results contradict an alternative model of S1P complexed with S1P 4 that suggested interactions predominantly in the extracellular loops [98]. Further modeling and mutagenesis studies identified W4.64 and K5.38 as positions that show variable importance in the S1P receptors [93,101]. Investigations of both LPA and S1P receptor indicate that a cationic residue in TM7 is often, but not universally, involved in ligand recognition [40,88,93].…”