Limits...
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.

Show MeSH

Related in: MedlinePlus

ITC analysis of 2-OG binding to the Af-GlnK3 variants F86I and F86P at 30°C.A) With respect to the wild type, the F86I variant shows reduced anticooperativity. The population microstate analysis (bottom panel) reveals that initially the singly-occupied species (▴) is populated, but that the negative cooperativity then is weaker so that the state with two bound ligands (▪) does accumulate before it yields to the fully occupied state (•) around a molar ratio of protein vs. ligand of 3. B) This effect is further enhanced in the F86P variant, where cooperativity is hardly seen in the microstate analysis and the binding sites are occupied sequentially. However, unlike in Af-GlnK2 that natively has P86, 2-OG is still bound.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3198391&req=5

pone-0026327-g007: ITC analysis of 2-OG binding to the Af-GlnK3 variants F86I and F86P at 30°C.A) With respect to the wild type, the F86I variant shows reduced anticooperativity. The population microstate analysis (bottom panel) reveals that initially the singly-occupied species (▴) is populated, but that the negative cooperativity then is weaker so that the state with two bound ligands (▪) does accumulate before it yields to the fully occupied state (•) around a molar ratio of protein vs. ligand of 3. B) This effect is further enhanced in the F86P variant, where cooperativity is hardly seen in the microstate analysis and the binding sites are occupied sequentially. However, unlike in Af-GlnK2 that natively has P86, 2-OG is still bound.

Mentions: As evidenced by the ITC analysis (Fig. 7) both Af-GlnK3 variants retained the ability to bind 2-OG, and in both cases this binding still showed cooperative behavior. The two Af-GlnK2 variants however, persisted in their incapacity to bind 2-OG under all tested conditions. Although this disproved our initial hypothesis that residue 86 might be the key to 2-OG binding and cooperativity, the analysis of population microstates did reveal significant differences with respect to wild type Af-GlnK3. In the F86I variant, the degree of negative cooperativity in 2-OG binding is reduced (Fig. 7A). The population with two bound ligands – virtually undetectable in the wild type (Fig. 6A) – is significantly populated. In the F86P protein this effect is even more pronounced, to the point that the three sites are sequentially populated and their mutual influence is reduced to a minimum. Residue 86 in the B-loop is thus not essential for 2-OG binding, but it does play a role in tuning the degree of cooperativity that we observe in the different members of the PII family.


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)

ITC analysis of 2-OG binding to the Af-GlnK3 variants F86I and F86P at 30°C.A) With respect to the wild type, the F86I variant shows reduced anticooperativity. The population microstate analysis (bottom panel) reveals that initially the singly-occupied species (▴) is populated, but that the negative cooperativity then is weaker so that the state with two bound ligands (▪) does accumulate before it yields to the fully occupied state (•) around a molar ratio of protein vs. ligand of 3. B) This effect is further enhanced in the F86P variant, where cooperativity is hardly seen in the microstate analysis and the binding sites are occupied sequentially. However, unlike in Af-GlnK2 that natively has P86, 2-OG is still bound.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026327-g007: ITC analysis of 2-OG binding to the Af-GlnK3 variants F86I and F86P at 30°C.A) With respect to the wild type, the F86I variant shows reduced anticooperativity. The population microstate analysis (bottom panel) reveals that initially the singly-occupied species (▴) is populated, but that the negative cooperativity then is weaker so that the state with two bound ligands (▪) does accumulate before it yields to the fully occupied state (•) around a molar ratio of protein vs. ligand of 3. B) This effect is further enhanced in the F86P variant, where cooperativity is hardly seen in the microstate analysis and the binding sites are occupied sequentially. However, unlike in Af-GlnK2 that natively has P86, 2-OG is still bound.
Mentions: As evidenced by the ITC analysis (Fig. 7) both Af-GlnK3 variants retained the ability to bind 2-OG, and in both cases this binding still showed cooperative behavior. The two Af-GlnK2 variants however, persisted in their incapacity to bind 2-OG under all tested conditions. Although this disproved our initial hypothesis that residue 86 might be the key to 2-OG binding and cooperativity, the analysis of population microstates did reveal significant differences with respect to wild type Af-GlnK3. In the F86I variant, the degree of negative cooperativity in 2-OG binding is reduced (Fig. 7A). The population with two bound ligands – virtually undetectable in the wild type (Fig. 6A) – is significantly populated. In the F86P protein this effect is even more pronounced, to the point that the three sites are sequentially populated and their mutual influence is reduced to a minimum. Residue 86 in the B-loop is thus not essential for 2-OG binding, but it does play a role in tuning the degree of cooperativity that we observe in the different members of the PII family.

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