Crystal Structure of a Complex between the Phosphorelay Protein YPD1 and the Response Regulator Domain of SLN1 Bound to a Phosphoryl Analog

The crystal structure of the yeast SLN1 response regulator (RR) domain bound to both a phosphoryl analog [beryllium fluoride (BeF3 −)] and Mg2 +, in complex with its downstream phosphorelay signaling partner YPD1, has been determined at a resolution of 1.70 Å. Comparisons between the BeF3 −-activated complex and the unliganded (or apo) complex determined previously reveal modest but important differences. The SLN1-R1·Mg2 +·BeF3 − structure from the complex provides evidence for the first time that the mechanism of phosphorylation-induced activation is highly conserved between bacterial RR domains and this example from a eukaryotic organism. Residues in and around the active site undergo slight rearrangements in order to form bonds with the essential divalent cation and fluorine atoms of BeF3 −. Two conserved switch-like residues (Thr1173 and Phe1192) occupy distinctly different positions in the apo versus BeF3 −-bound structures, consistent with the “Y–T” coupling mechanism proposed for the activation of CheY and other bacterial RRs. Several loop regions and the α4–β5–α5 surface of the SLN1-R1 domain undergo subtle conformational changes (∼ 1–3 Å displacements relative to the apo structure) that lead to significant changes in terms of contacts that are formed with YPD1. Detailed structural comparisons of protein–protein interactions in the apo and BeF3 −-bound complexes suggest at least a two-state equilibrium model for the formation of a transient encounter complex, in which phosphorylation of the RR promotes the formation of a phosphotransfer-competent complex. In the BeF3 −-activated complex, the position of His64 from YPD1 needs to be within ideal distance of and in near-linear geometry with Asp1144 from the SLN1-R1 domain for phosphotransfer to occur. The ground-state structure presented here suggests that phosphoryl transfer will likely proceed through an associative mechanism involving the formation of a pentacoordinate phosphorus intermediate.