Modulation of Cu²⁺ Binding to Sphingosine-1-Phosphate by Lipid Charge

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that is thought to participate in the regulation of many physiological processes and may play a key role in several diseases. Herein, we found that Cu²⁺ binds tightly to supported lipid bilayers (SLBs) containing S1P. Specifically, we demonstrated via fluorescence assays that Cu²⁺-S1P binding was bivalent and sensitive to the concentration of S1P in the SLB. In fact, the apparent equilibrium dissociation constant, K_DApp, tightened by a factor of 132 from 4.5 μM to 34 nM as the S1P density was increased from 5.0 to 20 mol %. A major driving force for this apparent tightening was the more negative surface potential with increasing S1P concentration. This potential remained unaltered upon Cu²⁺ binding at pH 7.4 because two protons were released for every Cu²⁺ that bound. At pH 5.4, however, Cu²⁺ could not outcompete protons for the amine and no binding occurred. Moreover, at pH 9.4, the amine was partially deprotonated before Cu²⁺ binding and the surface potential became more positive on binding. The results for Cu²⁺-S1P binding were reminiscent of those for Cu²⁺-phosphatidylserine binding, where a carboxylate group helped to deprotonate the amine. In the case of S1P, however, the phosphate needed to bear two negative charges to facilitate amine deprotonation in the presence of Cu²⁺.