Role of the highly conserved G68 residue in the yeast phosphorelay protein Ypd1: implications for interactions between histidine phosphotransfer (HPt) and response regulator proteins

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.Electronic supplementary materialThe online version of this article (10.1186/s12858-019-0104-5) contains supplementary material, which is available to authorized users.

Modeling of Protein–Protein Interactions in Cytokinin Signal Transduction

“…The yeast MSP system consists, similarly to plants, of three components: SLN1 is a sensor hybrid HK, YPD1 is a phosphorelay intermediate, and SSK1 is a response regulator. Three types of yeast protein dimers are available in the PDB: YPD1 in complex with SLN1 receiver domain [25,26], YPD1 homodimer [27], and YPD1 in complex with SSK1 [28]. More detailed information about the available structures of MSP and TCS components and their complexes can also be found in recent reviews [7,29].…”

Insights revealed by the co‐crystal structure of the Saccharomyces cerevisiae histidine phosphotransfer protein Ypd1 and the receiver domain of its downstream response regulator Ssk1

“…Removal of the nearby H637 imidazole ring produced a similar decrease in affinity (West lab, H637A, data not shown). The observed dissociation constants with Ypd1‐T12C∼F were calculated to be in the nM range for all Ssk1‐R2 variants, 2‐ to 10‐fold lower than the estimated dissociation constant for the Ypd1/Sln1‐R1 interaction . However, interaction between W638 (and/or H637) and the 5‐IAF fluorophore likely increased the apparent affinity between Ssk1‐R2 and labeled Ypd1, and a closer estimate of an absolute K d may be higher.…”

“…To confirm the estimates, an in vitro fluorescence‐binding assay was used to measure relative binding affinities between Ypd1 and Ssk1‐R2 variants that we created to disrupt these interactions . A threonine residue (T12) near the binding surface of Ypd1 was substituted with cysteine (T12C) and labeled with 5‐iodoacetamidofluorescein (5‐IAF).…”

“…A fluoresceinated Ypd1‐T12C variant (Ypd1‐T12C∼F) was created and used to test binding affinities with Ssk1‐R2 variants as previously described . Briefly, proteins were thawed and equilibrated (23°C) before each use.…”

“…Curves were converted to fractional fluorescence ( F / F 0 ) and shifted to begin at the origin for comparison purposes (( F / F 0 – 1) for Ssk1‐R2‐W638A; (1 – F/F 0 ) for all other Ssk1‐R2 variants). GraphPad Prism (v.8) was used to fit the data using an expanded quadratic equation to account for fluorescence from both bound and unbound states of Ypd1 . ΔΔ G binding values for each variant were determined using the relationship described by .…”