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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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Structural differences between (A) the ATP:Mg2+:2-OG complex and (B) the ADP complex of Af-GlnK3.In (A) the key ligand 2-oxolutarate requires the presence of ATP for binding and is located at the base of the T-loop (blue), with its ã-carboxy group forming a hydrogen bond to the conserved K58. Residue Q39 is the only protein ligand to the Mg2+ ion (grey sphere), and it is this residue that in the ADP complex (B) attains the exact position of 2-OG in (A), forming an analogous hydrogen bond to K58. The resulting tilt and shift of the base of the T-loop leads to a stable â-hairpin structure in (A), compared to a less well-ordered loop in (B) that moves inward by 20°towards the trimer. In both structures, the respective other T-loop conformation is indicated.
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pone-0026327-g002: Structural differences between (A) the ATP:Mg2+:2-OG complex and (B) the ADP complex of Af-GlnK3.In (A) the key ligand 2-oxolutarate requires the presence of ATP for binding and is located at the base of the T-loop (blue), with its ã-carboxy group forming a hydrogen bond to the conserved K58. Residue Q39 is the only protein ligand to the Mg2+ ion (grey sphere), and it is this residue that in the ADP complex (B) attains the exact position of 2-OG in (A), forming an analogous hydrogen bond to K58. The resulting tilt and shift of the base of the T-loop leads to a stable â-hairpin structure in (A), compared to a less well-ordered loop in (B) that moves inward by 20°towards the trimer. In both structures, the respective other T-loop conformation is indicated.

Mentions: A decrease of the cytoplasmic concentration of 2-OG is indicative of either a low carbon status (depletion through kataplerotic reactions) or of a high nitrogen status (conversion to glutamate/glutamine) [28]. In both cases Amt-mediated import of ammonium is no longer desired. Af-GlnK3 will return to the ATP-bound state, but will not yet form an inhibitory complex with Af-Amt3. Ammonium uptake will continue without negative effects on the cell, unless the energy level of the cell, expressed in the ratio ATP/ADP, starts to drop. At this stage the nucleotide diphosphate will replace ATP as a ligand of the PII protein, and it is this switch that gives the trimeric regulator the competence to bind tightly to Af-Amt3 and block transport. Energetic considerations strongly suggest the uptake of ammonium by Amt proteins to be an active mechanism driven by the proton motive force [1], [2]. At the same time, the intracellular accumulation of ammonium is unwanted, as the passive efflux of uncharged ammonia (that is in a protonation equilibrium with ammonium with a pKa of 9.25) would create a futile cycle to degrade the proton gradient [33], [34]. Ammonium is thus swiftly incorporated into glutamate or glutamine, at the expense of one molecule of NADPH or ATP, respectively. In a low energy situation, nitrogen is not required for growth, high-energy metabolites are scarce and the accumulation of intracellular ammonium places further stress on the proton motive force. Consequently, if ATP levels are too low to displace ADP from the GlnK protein, it efficiently shuts off ammonium uptake. In the structure of Af-GlnK3 with ADP, key residue Gln 39 was found to point inward to form a short (2.8 Å) hydrogen bond with the side chain of Lys 58 above the nucleotide. At the same time Glu 38 and Lys 101 form a salt bridge at the outward end of the nucleotide binding pocket and Phe 86 in the B-loop closes the remaining gap, effectively sealing up the nucleotide diphosphate within the Af-GlnK3 trimer (Fig. 2B). No Mg2+ ion was identified in the nucleotide binding pocket in this structure, and the overall conformation was very similar to that of Escherichia coli GlnK when bound to the ammonium transporter AmtB [27]. Consequently, this state of Af-GlnK3 is the one that is competent to bind to its transporter, Af-Amt3.


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)

Structural differences between (A) the ATP:Mg2+:2-OG complex and (B) the ADP complex of Af-GlnK3.In (A) the key ligand 2-oxolutarate requires the presence of ATP for binding and is located at the base of the T-loop (blue), with its ã-carboxy group forming a hydrogen bond to the conserved K58. Residue Q39 is the only protein ligand to the Mg2+ ion (grey sphere), and it is this residue that in the ADP complex (B) attains the exact position of 2-OG in (A), forming an analogous hydrogen bond to K58. The resulting tilt and shift of the base of the T-loop leads to a stable â-hairpin structure in (A), compared to a less well-ordered loop in (B) that moves inward by 20°towards the trimer. In both structures, the respective other T-loop conformation is indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198391&req=5

pone-0026327-g002: Structural differences between (A) the ATP:Mg2+:2-OG complex and (B) the ADP complex of Af-GlnK3.In (A) the key ligand 2-oxolutarate requires the presence of ATP for binding and is located at the base of the T-loop (blue), with its ã-carboxy group forming a hydrogen bond to the conserved K58. Residue Q39 is the only protein ligand to the Mg2+ ion (grey sphere), and it is this residue that in the ADP complex (B) attains the exact position of 2-OG in (A), forming an analogous hydrogen bond to K58. The resulting tilt and shift of the base of the T-loop leads to a stable â-hairpin structure in (A), compared to a less well-ordered loop in (B) that moves inward by 20°towards the trimer. In both structures, the respective other T-loop conformation is indicated.
Mentions: A decrease of the cytoplasmic concentration of 2-OG is indicative of either a low carbon status (depletion through kataplerotic reactions) or of a high nitrogen status (conversion to glutamate/glutamine) [28]. In both cases Amt-mediated import of ammonium is no longer desired. Af-GlnK3 will return to the ATP-bound state, but will not yet form an inhibitory complex with Af-Amt3. Ammonium uptake will continue without negative effects on the cell, unless the energy level of the cell, expressed in the ratio ATP/ADP, starts to drop. At this stage the nucleotide diphosphate will replace ATP as a ligand of the PII protein, and it is this switch that gives the trimeric regulator the competence to bind tightly to Af-Amt3 and block transport. Energetic considerations strongly suggest the uptake of ammonium by Amt proteins to be an active mechanism driven by the proton motive force [1], [2]. At the same time, the intracellular accumulation of ammonium is unwanted, as the passive efflux of uncharged ammonia (that is in a protonation equilibrium with ammonium with a pKa of 9.25) would create a futile cycle to degrade the proton gradient [33], [34]. Ammonium is thus swiftly incorporated into glutamate or glutamine, at the expense of one molecule of NADPH or ATP, respectively. In a low energy situation, nitrogen is not required for growth, high-energy metabolites are scarce and the accumulation of intracellular ammonium places further stress on the proton motive force. Consequently, if ATP levels are too low to displace ADP from the GlnK protein, it efficiently shuts off ammonium uptake. In the structure of Af-GlnK3 with ADP, key residue Gln 39 was found to point inward to form a short (2.8 Å) hydrogen bond with the side chain of Lys 58 above the nucleotide. At the same time Glu 38 and Lys 101 form a salt bridge at the outward end of the nucleotide binding pocket and Phe 86 in the B-loop closes the remaining gap, effectively sealing up the nucleotide diphosphate within the Af-GlnK3 trimer (Fig. 2B). No Mg2+ ion was identified in the nucleotide binding pocket in this structure, and the overall conformation was very similar to that of Escherichia coli GlnK when bound to the ammonium transporter AmtB [27]. Consequently, this state of Af-GlnK3 is the one that is competent to bind to its transporter, Af-Amt3.

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