Molecular Basis of Sphingosine Kinase 1 Substrate Recognition and Catalysis

Sphingosine kinase 1 (SphK1) is a lipid kinase that catalyzes the conversion of sphingosine to sphingosine-1-phosphate (S1P), which has been shown to play a role in lymphocyte trafficking, angiogenesis, and response to apoptotic stimuli. As a central enzyme in modulating the S1P levels in cells, SphK1 emerges as an important regulator for diverse cellular functions and a potential target for drug discovery. Here, we present the crystal structures of human SphK1 in the apo form and in complexes with a substrate sphingosine-like lipid, ADP, and an inhibitor at 2.0–2.3 Å resolution. The SphK1 structures reveal a two-domain architecture in which its catalytic site is located in the cleft between the two domains and a hydrophobic lipid-binding pocket is buried in the C-terminal domain. Comparative analysis of these structures with mutagenesis and kinetic studies provides insight into how SphK1 recognizes the lipid substrate and catalyzes ATP-dependent phosphorylation. ► SphK1 3D structure bears no similarity to protein kinases or other lipid kinases ► Lipid substrate is recognized by a deep cavity gated by a helix-loop-helix flap ► Asp81 is a key catalytic residue that facilitates direct phosphoryl transfer ► A reported SphK1 inhibitor binds competitively with substrate, not ATP Sphingosine kinase 1 (SphK1) is a lipid kinase central to modulating the S1P levels in cells. Wang et al. describe structures of human SphK1 that, together with mutagenesis and kinetic studies, reveal how SphK1 recognizes the lipid substrate, catalyzes ATPdependent phosphorylation, and permits inhibitor binding.