Extended N-terminal region of the essential phosphorelay signaling protein Ypd1 from Cryptococcus neoformans contributes to structural stability, phosphostability and binding of calcium ions

Abstract

Rapid response to external stimuli is crucial for survival and proliferation of microorganisms. Pathogenic fungi employ histidine-to-aspartate multistep phosphorelay systems to respond to environmental stress, progress through developmental stages and to produce virulence factors. Because these His-to-Asp phosphorelay systems are not found in humans, they are potential targets for the development of new antifungal therapies. Here we report the characterization of the histidine phosphotransfer (HPt) protein Ypd1 from the human fungal pathogen Cryptococcus neoformans Results from this study demonstrate that CnYpd1 indeed functions as a phosphorelay protein in vitro, and that H138 is confirmed as the site of phosphorylation. We found that CnYpd1 exhibits unique characteristics in comparison to other histidine phosphotransfer proteins, such as an extended N-terminal amino acid sequence, which we find contributes to structural integrity, a longer phosphorylated life time and the ability to bind calcium ions.

Keywords: Cryptococcus neoformans; His-to-Asp phosphorelay systems; Histidine-containing (HPt) phosphotransfer protein; Ypd1; signal transduction; two-component system.

Figure 1.

Representative sequence alignment of HPt proteins. Amino acid sequences of HPt proteins were aligned using ClustalX 2.1 and visualized using Jalview 2.8.2. Highly conserved residues are shaded dark blue, while less conserved residues are shaded in lighter blue. The phospho-accepting histidine residue is denoted by a red asterisk (*). Secondary structure of the known crystal structure of Ypd1 from S. cerevisiae (PDB ID: 1QSP) is diagrammed above the alignment.

Figure 2.

Phosphorylation of CnYpd1 from a heterologous phosphodonor. The HK and RR domains from a heterologous donor, Sln1 from S. cerevisiae (Sln1-HKR1), were used to phosphorylate CnYpd1. ScSln1-HKR1 was autophosphorylated using 0.1 μM γ-³²P-labeled ATP (lane 1). ScSln1-HKR1 was incubated with CnYpd1 alone (lane 2) or with ScSsk1-R2 (lane 3), CnYpd1-H138Q alone (lane 4) or with ScSsk1-R2 (lane 5).

Figure 3.

CnYpd1 N-terminal deletion mutants. Secondary structure predictions were performed using PSIPRED. Deletion mutants were created based on position of helices and region predicted to be an HPt domain.

Figure 4.

Extended phosphorylated life time of CnYpd1. The histidine kinase and receiver domains from the heterologous donor ScSln1 (ScSln1-HKR1) were used as a phospho-donor for CnYpd1. Phosphorylated ScSln1-HKR1 was removed from the reaction and aliquots of CnYpd1 were taken at specified time points. Three replicates were performed.

Figure 5.

CnYpd1 ΔN70 dephosphorylation. CnYpd1 ΔN70 was incubated with phospho∼GST-ScSln1-HKR1. GST-ScSln1-HKR1 was removed from the reaction. Aliquots were removed and mixed with stop buffer to quench the reaction. (A) All data points for phosphorylated half-life experiments as performed in quadruplicate for CnYpd1 ΔN70. Each colored line on the plot represents a separate experiment. (B) Representative phosphorimage that shows dephosphorylation of CnYpd1 ΔN70 over time. A phosphorylated half-life of 3.4 ± 0.63 h (N = 4) was calculated.

Figure 6.

Phosphorylation of CnYpd1, CnYpd1 E58A and CnYpd1 D60A-E67A. Phospho∼ScSln1-HKR1 was used as a donor to CnYpd1 constructs and removed from the reaction after 2 min. (A) Autophosphorylation of bead-bound ScSln1-HKR1 was performed in the presence of 10 mM MgCl₂. Bead-bound ScSln1-HKR1 was split into two fractions and each was washed by gentle pelleting of resin and addition of reaction buffer with either 10 mM MgCl₂ or CaCl₂. CnYpd1 was added to each ScSln1-HKR1 in the presence of CaCl₂(A) or MgCl₂(B) containing tube, ScSln1-HKR1 was removed from the reaction after 1 min and aliquots were taken of only CnYpd1 at 1, 5 and 15 min (lane designations indicate time points). (C) CnYpd1 E58A and CnYpd1 D60A-E67A were phosphorylated using ScSln1-HKR1. ScSln1-HKR1 was removed from the reaction after 1 min and aliquots were taken. Lane 1: WT CnYpd1, Lane 2: CnYpd1 E58A and lane 3: CnYpd1 D60A- E67A.

Figure 7.

Phylogenetic tree of species that encode HPt proteins with extended N-terminal region. Different species with stand-alone HPt proteins which exhibit an extended N-terminal region were used to generate a phylogenetic tree. Escherichia coli was used as an outgroup and is designated in gray. Purple represents the Fungi kingdom, orange the phylum Zygomycota, green Basidiomycota and blue Ascomycota. Pathogenic organisms are denoted by a asterisk sign and extremophiles by a plus sign.