Abstract
BackgroundMany bacteria and certain eukaryotes utilize multi-step His-to-Asp phosphorelays for adaptive responses to their extracellular environments. Histidine phosphotransfer (HPt) proteins function as key components of these pathways. HPt proteins are genetically diverse, but share a common tertiary fold with conserved residues near the active site. A surface-exposed glycine at the H + 4 position relative to the phosphorylatable histidine is found in a significant number of annotated HPt protein sequences. Previous reports demonstrated that substitutions at this position result in diminished phosphotransfer activity between HPt proteins and their cognate signaling partners.ResultsWe report the analysis of partner binding interactions and phosphotransfer activity of the prototypical HPt protein Ypd1 from Saccharomyces cerevisiae using a set of H + 4 (G68) substituted proteins. Substitutions at this position with large, hydrophobic, or charged amino acids nearly abolished phospho-acceptance from the receiver domain of its upstream signaling partner, Sln1 (Sln1-R1). An in vitro binding assay indicated that G68 substitutions caused only modest decreases in affinity between Ypd1 and Sln1-R1, and these differences did not appear to be large enough to account for the observed decrease in phosphotransfer activity. The crystal structure of one of these H + 4 mutants, Ypd1-G68Q, which exhibited a diminished ability to participate in phosphotransfer, shows a similar overall structure to that of wild-type. Molecular modelling suggests that the highly conserved active site residues within the receiver domain of Sln1 must undergo rearrangement to accommodate larger H + 4 substitutions in Ypd1.ConclusionsPhosphotransfer reactions require precise arrangement of active site elements to align the donor-acceptor atoms and stabilize the transition state during the reaction. Any changes likely result in an inability to form a viable transition state during phosphotransfer. Our data suggest that the high degree of evolutionary conservation of residues with small side chains at the H + 4 position in HPt proteins is required for optimal activity and that the presence of larger residues at the H + 4 position would cause alterations in the positioning of active site residues in the partner response regulator.
Highlights
Many bacteria and certain eukaryotes utilize multi-step His-to-Asp phosphorelays for adaptive responses to their extracellular environments
Residues in the H + 4 position of Histidine phosphotransfer (HPt) domains Approximately 10,000 non-redundant sequences, representing bacteria, fungi, and plant HPt sequences obtained from the Pfam database [49], were analyzed to determine what percentage of residues were found in the H + 4 position
Non-conservative substitutions at the H + 4 position disrupt phosphotransfer Various amino acid residues (S, A, V, L, E, Q) were introduced to the G68 position of Ypd1 in order to analyze what effects amino acids other than glycine have on the phosphorelay activity of Ypd1
Summary
Many bacteria and certain eukaryotes utilize multi-step His-to-Asp phosphorelays for adaptive responses to their extracellular environments. Histidine phosphotransfer (HPt) proteins allow bacteria, yeast, and plants to expand beyond canonical two-component signaling pathways into multi-step phosphorelay pathways. These signaling proteins play crucial roles in regulating numerous cellular functions including those essential for growth and viability [1,2,3,4]. Multi-step phosphorelay systems consist of an upstream hybrid sensor histidine kinase (HHK), an HPt protein, and often multiple downstream response regulators (RR). The HPt protein (or domain) acts as an intermediate in the signaling pathway by transferring the phosphoryl group from the HHK to a RR, which elicits a cellular response to the detected stress [1, 15, 16]
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