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. 2009 Dec 8;48(48):11522-31.
doi: 10.1021/bi9011594.

Sensation and signaling of alpha-ketoglutarate and adenylylate energy charge by the Escherichia coli PII signal transduction protein require cooperation of the three ligand-binding sites within the PII trimer

Peng Jiang  1 , Alexander J Ninfa
Affiliations

Sensation and signaling of alpha-ketoglutarate and adenylylate energy charge by the Escherichia coli PII signal transduction protein require cooperation of the three ligand-binding sites within the PII trimer

Peng Jiang et al. Biochemistry. .

Abstract

PII proteins are sensors of alpha-ketoglutarate and adenylylate energy charge that regulate signal transduction proteins, metabolic enzymes, and permeases involved in nitrogen assimilation. Here, purified Escherichia coli PII and two of its receptors, ATase and NRII, were used to study the mechanisms of sensation by PII. We assembled heterotrimeric forms of PII from wild-type and mutant subunits, which allowed us to assess the role of the three binding sites for alpha-ketoglutarate and adenylylate nucleotide in the PII trimer. Signaling of alpha-ketoglutarate and adenylylate energy charge by these heterotrimeric PII proteins required multiple binding sites for these effectors, and the ligand-binding sites on different subunits could influence the function of a single subunit interacting with a receptor, implying communication between PII subunits. Wild-type and heterotrimeric forms of PII were also used to examine the effects of alpha-ketoglutarate and ADP on PII activation of the adenylyltransferase (AT) activity of ATase. Previous work showed that when ATP was the sole adenylylate nucleotide, alpha-ketoglutarate controlled the extent of PII activation but did not alter the PII activation constant (K(act)). We show that ADP affected both the PII K(act) and the extent of activation by PII. When ATP was present, ADP dramatically reduced the K(act) for wild-type PII, and this effect was antagonized by alpha-ketoglutarate. Consequently, when ATP was present, the antagonism between ADP and alpha-ketoglutarate allowed each of these effectors to influence the PII K(act) for activation of ATase. A study of heterotrimeric forms of PII suggested that the major part of the ability of ADP to improve the binding of PII to ATase required multiple nucleotide binding sites and intersubunit communication. We also used nondenaturing gel electrophoresis to investigate the effect of ADP and alpha-ketoglutarate on the binding of PII to ATase and NRII. These studies showed that ATase and NRII differ in their requirements for interaction with PII, and that under the appropriate conditions, the antagonism between alpha-ketoglutarate and ADP allowed each of these effectors to influence the binding of PII to receptors.

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Figures

Figure 1
Figure 1
Sensation and signaling of α-ketoglutarate by heterotrimeric forms of PII. Initial rates of GS adenylylation by ATase (AT activity) were measured as described in Materials and Methods. Conditions were 2.5 μM GS, 0.1 μM ATase, 0.5 mM [α-32P]ATP, PII heterotrimers with the concentration of wild-type subunits at 3 μM, and α-ketoglutarate as indicated: (●) heterotrimers containing one wild-type subunit and two Δ47−53 subunits and (◼) heterotrimers containing one wild-type subunit and two G89A subunits.
Figure 2
Figure 2
Sensation and signaling of adenylylate energy charge and α-ketoglutarate by wild-type and heterotrimeric forms of PII. The initial rate of GS adenylylation by ATase (AT activity) was measured as described in Materials and Methods. (A) Signaling of decreasing adenylylate energy charge at a low (0.03 mM) α-ketoglutarate concentration. Decreasing adenylylate energy charge was caused by addition of ADP to reaction mixtures with a fixed concentration of ATP. Conditions were 2.5 μM GS, 0.5 mM [α-32P]ATP, 0.1 μM ATase, 1.5 μM wild-type PII subunits, 0.03 mM α-ketoglutarate, and ADP as indicated: (white bars) wild-type PII, (hatched bars) heterotrimers containing one wild-type subunit and two Δ47−53 subunits, and (gray bars) heterotrimers containing one wild-type subunit and two G89A subunits. (B) Signaling of decreasing adenylylate energy charge at a high (1 mM) α-ketoglutarate concentration. Conditions and symbols are as for panel A, except that the concentration of ATase was doubled (to 0.2 μM) to partially compensate for the low AT activity under these conditions.
Figure 3
Figure 3
PII activation constants (Kact) for activation of the AT activity of ATase. The initial rate of GS adenylylation by ATase (AT activity) was measured as described in Materials and Methods, with 2.5 μM GS, 0.1 μM ATase, 0.5 mM [α-32P]ATP, α-ketoglutarate at 0.05 or 1.0 mM, as indicated, and PII as indicated. As noted in Materials and Methods, the PII heterotrimer samples used were a mixture of species, consisting mainly of the innocuous mutant homotrimers along with the heterotrimers. The concentration of PII heterotrimers (X-axis) is expressed as the concentration of wild-type PII trimers used to assemble the quantity of heterotrimers (i.e., one-third the concentration of the wild-type subunits). This allows a comparison of the activity and activation constant of the heterotrimer samples with the published activity and activation constant of wild-type PII homotrimers. (A) Activation by heterotrimers containing G89A subunits, at a low α-ketoglutarate concentration. (B) Activation by heterotrimers containing Δ47−53 subunits, at a low α-ketoglutarate concentration. (C) Activation by heterotrimers containing G89A subunits, at a high α-ketoglutarate concentration. (D) Activation by heterotrimers containing Δ47−53 subunits, at a high α-ketoglutarate concentration.
Figure 4
Figure 4
PII activation constants (Kact) for activation of the AT activity of ATase in the presence of ADP. The initial rate of GS adenylylation by ATase (AT activity) was measured as described in Materials and Methods, with 2.5 μM GS, 0.5 mM [α-32P]ATP, and the indicated concentrations of ATase, α-ketoglutarate, ADP, and PII. The concentration of PII is expressed as the homotrimer concentration, while the concentration of the heterotrimeric species is expressed as the concentration of wild-type PII trimers used to assemble the quantity of heterotrimers (i.e., one-third the concentration of the wild-type subunits). (A) Activation by wild-type PII homotrimers. Conditions were 0.025 μM ATase, 0.05 mM α-ketoglutarate, and 1 mM ADP. (B) Activation by wild-type PII homotrimers. Conditions were 0.08 μM ATase, 1 mM ADP, and 1 mM α-ketoglutarate. (C) Activation by heterotrimers containing G89A subunits. Conditions were 0.1 μM ATase, 0.05 mM α-ketoglutarate, and 1 mM ADP. (D) Activation by PII heterotrimers containing Δ47−53 subunits. Conditions were 0.1 μM ATase, 0.05 mM α-ketoglutarate, and 1 mM ADP.
Figure 5
Figure 5
Nondenaturing gel electrophoresis analysis of the binding of PII to ATase and NRII. Procedures were as in Materials and Methods; the concentrations of proteins in the initial incubation mixtures are shown above each gel lane. A constant volume of each sample was loaded, such that the proportions of proteins in the gel parallel their initial concentrations. (A) Reaction mixtures, gel, and running buffer contained 0.5 mM ATP and lacked α-ketoglutarate and ADP. (B) Reaction mixtures, gel, and running buffer contained 0.5 mM ADP and lacked ATP and α-ketoglutarate. (C) Reaction mixtures, gel, and running buffer contained 0.5 mM ADP and 0.03 mM α-ketoglutarate and lacked ATP. (D) Reaction mixtures, gel, and running buffer contained 0.5 mM ADP and 1 mM α-ketoglutarate and lacked ATP. The bands corresponding to PII, NRII, and ATase are indicated, and complexes are marked with an asterisk.
Figure 6
Figure 6
Hypothetical signaling states of PII. The PII trimer is schematically depicted as three linked circles. Black dots correspond to bound α-ketoglutarate molecules. T and D denote bound ATP and ADP, respectively. As shown above the dotted line, there are 16 different forms of PII in which all three nucleotide binding sites of the trimer are occupied and at least one molecule of α-ketoglutarate is bound. In all, there are 56 possible liganded states of the PII protein (species above and below the dotted line).
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