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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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Af-GlnK3 and its physiological role in ammonium uptake.A) Top view of the trimer Af-GlnK3, highlighting the ligand binding sites between the monomers and the protruding T-loops that are required for blocking ammonium transport. B) As discussed previously [1], [33], ammonium is actively taken up by Amt proteins and used to aminate glutamate in the ATP-dependent reaction of glutamine synthetase (GS). This reaction is coupled to glutamate∶oxoglutarate amidotransferase (GOGAT) that forms two molecules of glutamate from glutamine and 2-oxoglutarate.
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pone-0026327-g001: Af-GlnK3 and its physiological role in ammonium uptake.A) Top view of the trimer Af-GlnK3, highlighting the ligand binding sites between the monomers and the protruding T-loops that are required for blocking ammonium transport. B) As discussed previously [1], [33], ammonium is actively taken up by Amt proteins and used to aminate glutamate in the ATP-dependent reaction of glutamine synthetase (GS). This reaction is coupled to glutamate∶oxoglutarate amidotransferase (GOGAT) that forms two molecules of glutamate from glutamine and 2-oxoglutarate.

Mentions: Af-GlnK3 shares the canonical fold of PII proteins, consisting of a four-stranded, antiparallel beta sheet and two connecting alpha helices. It forms a tightly packed trimer with approximate dimensions of 30×54 Å (Fig. 1A). The crucial structural feature is the loop connecting beta strands 2 and 3, the T-loop. It spans 21 amino acid residues from G35 to L56 and undergoes conformational changes upon binding of effector molecules that directly affect the affinity of the protein for its interaction partner, Af-Amt3. The T-loop shows intrinsic flexibility that is key to its functionality, and in consequence this loop was disordered in most of the PII protein structures available to date [24]. In the present work, the thermostable ortholog Af-GlnK3 does allow for the observation of a defined conformation for the T-loop with the bound effectors MgATP and 2-OG (PDB code 3TA2) that prohibits binding to Af-Amt3, as well as with bound ADP (PDB code 3TA1), in a conformation that promotes this complex formation.


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)

Af-GlnK3 and its physiological role in ammonium uptake.A) Top view of the trimer Af-GlnK3, highlighting the ligand binding sites between the monomers and the protruding T-loops that are required for blocking ammonium transport. B) As discussed previously [1], [33], ammonium is actively taken up by Amt proteins and used to aminate glutamate in the ATP-dependent reaction of glutamine synthetase (GS). This reaction is coupled to glutamate∶oxoglutarate amidotransferase (GOGAT) that forms two molecules of glutamate from glutamine and 2-oxoglutarate.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026327-g001: Af-GlnK3 and its physiological role in ammonium uptake.A) Top view of the trimer Af-GlnK3, highlighting the ligand binding sites between the monomers and the protruding T-loops that are required for blocking ammonium transport. B) As discussed previously [1], [33], ammonium is actively taken up by Amt proteins and used to aminate glutamate in the ATP-dependent reaction of glutamine synthetase (GS). This reaction is coupled to glutamate∶oxoglutarate amidotransferase (GOGAT) that forms two molecules of glutamate from glutamine and 2-oxoglutarate.
Mentions: Af-GlnK3 shares the canonical fold of PII proteins, consisting of a four-stranded, antiparallel beta sheet and two connecting alpha helices. It forms a tightly packed trimer with approximate dimensions of 30×54 Å (Fig. 1A). The crucial structural feature is the loop connecting beta strands 2 and 3, the T-loop. It spans 21 amino acid residues from G35 to L56 and undergoes conformational changes upon binding of effector molecules that directly affect the affinity of the protein for its interaction partner, Af-Amt3. The T-loop shows intrinsic flexibility that is key to its functionality, and in consequence this loop was disordered in most of the PII protein structures available to date [24]. In the present work, the thermostable ortholog Af-GlnK3 does allow for the observation of a defined conformation for the T-loop with the bound effectors MgATP and 2-OG (PDB code 3TA2) that prohibits binding to Af-Amt3, as well as with bound ADP (PDB code 3TA1), in a conformation that promotes this complex formation.

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