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Mechanism of disruption of the Amt-GlnK complex by P(II)-mediated sensing of 2-oxoglutarate.

Maier S, Schleberger P, Lü W, Wacker T, Pflüger T, Litz C, Andrade SL - PLoS ONE (2011)

Bottom Line: Contrary to Af-GlnK2 this protein was able to bind both ATP/2-OG and ADP to yield inactive and functional states, respectively.Due to the thermostable nature of the protein we could observe the exact positioning of the notoriously flexible T-loops and explain the binding behavior of GlnK proteins to their interaction partner, the Amt proteins.A thermodynamic analysis of these binding events using microcalorimetry evaluated by microstate modeling revealed significant differences in binding cooperativity compared to other characterized P(II) proteins, underlining the diversity and adaptability of this class of regulatory signaling proteins.

View Article: PubMed Central - PubMed

Affiliation: Institut für organische Chemie und Biochemie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.

ABSTRACT
GlnK proteins regulate the active uptake of ammonium by Amt transport proteins by inserting their regulatory T-loops into the transport channels of the Amt trimer and physically blocking substrate passage. They sense the cellular nitrogen status through 2-oxoglutarate, and the energy level of the cell by binding both ATP and ADP with different affinities. The hyperthermophilic euryarchaeon Archaeoglobus fulgidus possesses three Amt proteins, each encoded in an operon with a GlnK ortholog. One of these proteins, GlnK2 was recently found to be incapable of binding 2-OG, and in order to understand the implications of this finding we conducted a detailed structural and functional analysis of a second GlnK protein from A. fulgidus, GlnK3. Contrary to Af-GlnK2 this protein was able to bind both ATP/2-OG and ADP to yield inactive and functional states, respectively. Due to the thermostable nature of the protein we could observe the exact positioning of the notoriously flexible T-loops and explain the binding behavior of GlnK proteins to their interaction partner, the Amt proteins. A thermodynamic analysis of these binding events using microcalorimetry evaluated by microstate modeling revealed significant differences in binding cooperativity compared to other characterized P(II) proteins, underlining the diversity and adaptability of this class of regulatory signaling proteins.

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2-OG binding to Af-GlnK3 and temperature dependence.A) A titration at 30°C shows an initial endothermic event indicating an entropy-driven process that is followed by a strongly exothermic, enthalpy-driven event. In the analysis of population microstates (bottom panel) this translates to an initial accumulation of the singly occupied species (▴) due to negative cooperativity for the second site (▪), but strong positive cooperativity for binding the third ligand (•). Only singly or fully occupied binding sites will be present in relevant amounts. B) At 70°C the initial binding event becomes exothermic, leading to a very different overall shape of the experimental curve (top panel). However, analysis of the population microstates shows the same qualitative behavior as in (A).
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pone-0026327-g006: 2-OG binding to Af-GlnK3 and temperature dependence.A) A titration at 30°C shows an initial endothermic event indicating an entropy-driven process that is followed by a strongly exothermic, enthalpy-driven event. In the analysis of population microstates (bottom panel) this translates to an initial accumulation of the singly occupied species (▴) due to negative cooperativity for the second site (▪), but strong positive cooperativity for binding the third ligand (•). Only singly or fully occupied binding sites will be present in relevant amounts. B) At 70°C the initial binding event becomes exothermic, leading to a very different overall shape of the experimental curve (top panel). However, analysis of the population microstates shows the same qualitative behavior as in (A).

Mentions: Af-GlnK3 bound MgATP and MgADP, and the binding of 2-OG required pre-incubation of the protein with MgATP, in line with data published on A. brasilense GlnZ, E. coli GlnK and S. elongatus PII [31], [32], [40], [41]. Binding of MgATP to Af-GlnK3 was roughly two-fold stronger than binding of MgADP, either at 30 or 70°C although the binding affinity for both nucleotide molecules is clearly higher at 30°C (Table 2). The effect of replacing the bulky F86 for isoleucine, a more common residue among the PII protein family, or proline (as it occurs in Af-GlnK2) resulted in a general increase in the nucleotide binding affinities. The F86I variant bound MgATP and MgADP with about 2-fold increased affinity when compared to the wild-type protein, but still displayed a 2-3 fold preference for MgATP binding. Similarly the F86P variant also showed higher binding constants for both nucleotides than the wild-type protein. The affinity constants for MgATP were identical for both variants, but Af-GlnK3 F86P showed 5–6-fold higher affinity to MgADP and 1–2 fold stronger binding of MgATP than the wild type (Table 2). The total Gibbs free energy calculations confirm that replacing F86 for an isoleucine resulted in a variant that bound MgATP and MgADP more favorably than the wild type by about 0.5 and 0.3 kcal.mol−1, while replacement with a proline resulted in 0.6 and 1.0 kcal.mol−1, respectively, changing the nucleotide preference in favor of ADP. When compared to Af-GlnK2, the observed affinities for the nucleotides were lower. More importantly, the distinct cooperative binding behavior of the trimeric molecule was absent. The heat developed during the titration experiments of Af-GlnK3 with MgATP and MgADP yielded data that could be fit with a single sigmoidal profile derived from a single-site model with three independent sites that did not show cooperativity (Fig. 5, Table 2). However, this was not the case when 2-OG was titrated to Af-GlnK3 with bound MgATP. Here the experimental curves showed a complex, cooperative binding scheme that could be fitted using a sequential binding model with three sites. An analysis of the population microstates for 2-OG binding provided the first detailed evidence for the negative cooperativity in this second ligand binding step that is generally assumed for GlnK proteins (Fig. 6) and that allows the proteins to sample a wide range of ligand concentrations.


Mechanism of disruption of the Amt-GlnK complex by P(II)-mediated sensing of 2-oxoglutarate.

Maier S, Schleberger P, Lü W, Wacker T, Pflüger T, Litz C, Andrade SL - PLoS ONE (2011)

2-OG binding to Af-GlnK3 and temperature dependence.A) A titration at 30°C shows an initial endothermic event indicating an entropy-driven process that is followed by a strongly exothermic, enthalpy-driven event. In the analysis of population microstates (bottom panel) this translates to an initial accumulation of the singly occupied species (▴) due to negative cooperativity for the second site (▪), but strong positive cooperativity for binding the third ligand (•). Only singly or fully occupied binding sites will be present in relevant amounts. B) At 70°C the initial binding event becomes exothermic, leading to a very different overall shape of the experimental curve (top panel). However, analysis of the population microstates shows the same qualitative behavior as in (A).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198391&req=5

pone-0026327-g006: 2-OG binding to Af-GlnK3 and temperature dependence.A) A titration at 30°C shows an initial endothermic event indicating an entropy-driven process that is followed by a strongly exothermic, enthalpy-driven event. In the analysis of population microstates (bottom panel) this translates to an initial accumulation of the singly occupied species (▴) due to negative cooperativity for the second site (▪), but strong positive cooperativity for binding the third ligand (•). Only singly or fully occupied binding sites will be present in relevant amounts. B) At 70°C the initial binding event becomes exothermic, leading to a very different overall shape of the experimental curve (top panel). However, analysis of the population microstates shows the same qualitative behavior as in (A).
Mentions: Af-GlnK3 bound MgATP and MgADP, and the binding of 2-OG required pre-incubation of the protein with MgATP, in line with data published on A. brasilense GlnZ, E. coli GlnK and S. elongatus PII [31], [32], [40], [41]. Binding of MgATP to Af-GlnK3 was roughly two-fold stronger than binding of MgADP, either at 30 or 70°C although the binding affinity for both nucleotide molecules is clearly higher at 30°C (Table 2). The effect of replacing the bulky F86 for isoleucine, a more common residue among the PII protein family, or proline (as it occurs in Af-GlnK2) resulted in a general increase in the nucleotide binding affinities. The F86I variant bound MgATP and MgADP with about 2-fold increased affinity when compared to the wild-type protein, but still displayed a 2-3 fold preference for MgATP binding. Similarly the F86P variant also showed higher binding constants for both nucleotides than the wild-type protein. The affinity constants for MgATP were identical for both variants, but Af-GlnK3 F86P showed 5–6-fold higher affinity to MgADP and 1–2 fold stronger binding of MgATP than the wild type (Table 2). The total Gibbs free energy calculations confirm that replacing F86 for an isoleucine resulted in a variant that bound MgATP and MgADP more favorably than the wild type by about 0.5 and 0.3 kcal.mol−1, while replacement with a proline resulted in 0.6 and 1.0 kcal.mol−1, respectively, changing the nucleotide preference in favor of ADP. When compared to Af-GlnK2, the observed affinities for the nucleotides were lower. More importantly, the distinct cooperative binding behavior of the trimeric molecule was absent. The heat developed during the titration experiments of Af-GlnK3 with MgATP and MgADP yielded data that could be fit with a single sigmoidal profile derived from a single-site model with three independent sites that did not show cooperativity (Fig. 5, Table 2). However, this was not the case when 2-OG was titrated to Af-GlnK3 with bound MgATP. Here the experimental curves showed a complex, cooperative binding scheme that could be fitted using a sequential binding model with three sites. An analysis of the population microstates for 2-OG binding provided the first detailed evidence for the negative cooperativity in this second ligand binding step that is generally assumed for GlnK proteins (Fig. 6) and that allows the proteins to sample a wide range of ligand concentrations.

Bottom Line: Contrary to Af-GlnK2 this protein was able to bind both ATP/2-OG and ADP to yield inactive and functional states, respectively.Due to the thermostable nature of the protein we could observe the exact positioning of the notoriously flexible T-loops and explain the binding behavior of GlnK proteins to their interaction partner, the Amt proteins.A thermodynamic analysis of these binding events using microcalorimetry evaluated by microstate modeling revealed significant differences in binding cooperativity compared to other characterized P(II) proteins, underlining the diversity and adaptability of this class of regulatory signaling proteins.

View Article: PubMed Central - PubMed

Affiliation: Institut für organische Chemie und Biochemie, Albert-Ludwigs-Universität Freiburg, Freiburg, Germany.

ABSTRACT
GlnK proteins regulate the active uptake of ammonium by Amt transport proteins by inserting their regulatory T-loops into the transport channels of the Amt trimer and physically blocking substrate passage. They sense the cellular nitrogen status through 2-oxoglutarate, and the energy level of the cell by binding both ATP and ADP with different affinities. The hyperthermophilic euryarchaeon Archaeoglobus fulgidus possesses three Amt proteins, each encoded in an operon with a GlnK ortholog. One of these proteins, GlnK2 was recently found to be incapable of binding 2-OG, and in order to understand the implications of this finding we conducted a detailed structural and functional analysis of a second GlnK protein from A. fulgidus, GlnK3. Contrary to Af-GlnK2 this protein was able to bind both ATP/2-OG and ADP to yield inactive and functional states, respectively. Due to the thermostable nature of the protein we could observe the exact positioning of the notoriously flexible T-loops and explain the binding behavior of GlnK proteins to their interaction partner, the Amt proteins. A thermodynamic analysis of these binding events using microcalorimetry evaluated by microstate modeling revealed significant differences in binding cooperativity compared to other characterized P(II) proteins, underlining the diversity and adaptability of this class of regulatory signaling proteins.

Show MeSH
Related in: MedlinePlus