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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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Binding mode of the ligands ATP, Mg2+ and 2-oxoglutarate to Af-GlnK3.The stereo image shows a view into the ligand-binding cleft located at the interface of two monomers, one of which (dark green) provides the T-loop (blue) and B-loop regions to the binding site, the other monomer (light green) the C-loop. The Mg2+ ion (grey sphere) shows octahedral coordination by all three phosphate groups of ATP, by the á-carboxy and á-keto functions of 2-oxoglutarate and by the ã-amido oxygen atom of residue Q39.
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pone-0026327-g003: Binding mode of the ligands ATP, Mg2+ and 2-oxoglutarate to Af-GlnK3.The stereo image shows a view into the ligand-binding cleft located at the interface of two monomers, one of which (dark green) provides the T-loop (blue) and B-loop regions to the binding site, the other monomer (light green) the C-loop. The Mg2+ ion (grey sphere) shows octahedral coordination by all three phosphate groups of ATP, by the á-carboxy and á-keto functions of 2-oxoglutarate and by the ã-amido oxygen atom of residue Q39.

Mentions: Although the binding modes of 2-OG in the three structures available to date are almost identical (and distinct from an earlier observation of a single 2-OG molecule bound to a very different position in Methanococcus jannaschii GlnK1) [36], the effect on the conformation of the T-loop is fundamentally different. In the structure of the PII protein from S. elongatus the loop is disordered [32], while in A. brasilense GlnZ it shows a defined conformation, but points away laterally from the disc-shaped trimer [31]. In Af-GlnK3 the T-loops shift to attain a highly ordered β-hairpin conformation stabilized by six hydrogen bonds involving peptide amides, and are fully ordered in the crystal structure (Fig. 2A, 3). Residue Arg 47 that is crucial for insertion into the substrate channels of the Amt protein upon complex formation, remains poised at the apex of the loop. However, 2-OG is fixed at the base of the loop in a wedge-like manner and pushes the T-loops outward with respect to their conformation in the ADP-bound state. In both structures, the Cα atoms of residue Arg 47 form an equilateral triangle, but while the sides of this triangle in a complex with the Amt protein have a length of 31 Å, they are extended to 46 Å in the form with bound 2-OG (Fig. 4). They thus lose their structural flexibility and are pried apart too far to be able to insert into the substrate exit channels of Af-Amt3. For A. brasilense GlnZ, whose direct interaction partner remains to be identified, there is likely no similarly strict requirement and its T-loops, although based on the identical ligand-binding mode, orient differently.


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)

Binding mode of the ligands ATP, Mg2+ and 2-oxoglutarate to Af-GlnK3.The stereo image shows a view into the ligand-binding cleft located at the interface of two monomers, one of which (dark green) provides the T-loop (blue) and B-loop regions to the binding site, the other monomer (light green) the C-loop. The Mg2+ ion (grey sphere) shows octahedral coordination by all three phosphate groups of ATP, by the á-carboxy and á-keto functions of 2-oxoglutarate and by the ã-amido oxygen atom of residue Q39.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026327-g003: Binding mode of the ligands ATP, Mg2+ and 2-oxoglutarate to Af-GlnK3.The stereo image shows a view into the ligand-binding cleft located at the interface of two monomers, one of which (dark green) provides the T-loop (blue) and B-loop regions to the binding site, the other monomer (light green) the C-loop. The Mg2+ ion (grey sphere) shows octahedral coordination by all three phosphate groups of ATP, by the á-carboxy and á-keto functions of 2-oxoglutarate and by the ã-amido oxygen atom of residue Q39.
Mentions: Although the binding modes of 2-OG in the three structures available to date are almost identical (and distinct from an earlier observation of a single 2-OG molecule bound to a very different position in Methanococcus jannaschii GlnK1) [36], the effect on the conformation of the T-loop is fundamentally different. In the structure of the PII protein from S. elongatus the loop is disordered [32], while in A. brasilense GlnZ it shows a defined conformation, but points away laterally from the disc-shaped trimer [31]. In Af-GlnK3 the T-loops shift to attain a highly ordered β-hairpin conformation stabilized by six hydrogen bonds involving peptide amides, and are fully ordered in the crystal structure (Fig. 2A, 3). Residue Arg 47 that is crucial for insertion into the substrate channels of the Amt protein upon complex formation, remains poised at the apex of the loop. However, 2-OG is fixed at the base of the loop in a wedge-like manner and pushes the T-loops outward with respect to their conformation in the ADP-bound state. In both structures, the Cα atoms of residue Arg 47 form an equilateral triangle, but while the sides of this triangle in a complex with the Amt protein have a length of 31 Å, they are extended to 46 Å in the form with bound 2-OG (Fig. 4). They thus lose their structural flexibility and are pried apart too far to be able to insert into the substrate exit channels of Af-Amt3. For A. brasilense GlnZ, whose direct interaction partner remains to be identified, there is likely no similarly strict requirement and its T-loops, although based on the identical ligand-binding mode, orient differently.

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