CBS domains form energy-sensing modules whose binding of adenosine ligands is disrupted by disease mutations (original) (raw)
The N-terminal and C-terminal CBS domain pairs from AMPK-γ2 bind one molecule of AMP or ATP. The crystal structure of a bacterial IMPDH shows that the tandem pair of CBS domains are intimately associated via hydrophobic interactions between homologous β sheets composed of three strands from one domain and one from the other (34), making it very unlikely that single domains would be stable. We therefore cloned DNA from human AMPK-γ2 encoding either all four domains (CBS1-4), or the N-terminal pair (CBS1-2) or the C-terminal pair (CBS3-4). All three constructs were expressed as GST fusions in bacteria and were purified on glutathione-Sepharose as soluble proteins of the expected sizes in good yield. Binding of AMP was assessed using [14C]AMP and a rapid-filtration method. Initial fitting of the data using the WT CBS1-2 fusion showed that the maximal binding was very close to 1 mole (0.95 ± 0.05) per mole of protein, so data were subsequently fitted to a single-site binding model (Figure 1a), yielding a dissociation constant (K_d) of 53 μM. We also used site-directed mutagenesis to create four of the mutations in CBS1-2 that cause WPWS, i.e., R302Q, H383R and T400N, and L_ins (which inserts a leucine residue in the linker between CBS1 and CBS2). All mutants still bound one molecule of AMP, but the _K_d values for R302Q, T400N, and H383R were increased six-, ten-, and 28-fold, respectively, relative to that for the WT (Figure 1a and Table 1). The K_d for L_ins was not significantly different from that for the WT.
Binding of AMP (a and c) or ATP (b and d) by the CBS1-2 construct (a and b) or the CBS3-4 construct (c and d). The fusion proteins were either the WT (open circles) or one of five point mutants (R302Q, filled circles; L_ins_, open squares; H383R, filled squares; T400N, open triangles; R531G, filled triangles). Data were fitted to a single-site binding model: bound = [nucleotide]/(_K_d + [nucleotide]). The curves are theoretical curves obtained using the _K_d values shown in Table 1.
Since high concentrations of ATP antagonize activation of AMPK by AMP (35), we suspected that the CBS1-2 proteins would also bind ATP. This was indeed the case, and for the WT the data could be fitted to a single-site binding model with a _K_d 3.3-fold higher than that for AMP. All of the mutants still bound ATP, but the K_d values were increased in the same rank order as for AMP binding, albeit to a lesser extent. For R302Q, T400N, and H383R, the increases were three-, five-, and ninefold, respectively, while L_ins was again not significantly different from the WT (Figure 1b and Table 1). High concentrations of ATP completely displaced binding of AMP from the WT (Figure 2), showing that binding of the two nucleotides is mutually exclusive. We also measured the apparent _K_d for AMP of the WT CBS1-2 protein in the presence of four different concentrations of ATP and fitted the results to the equation: apparent _K_d = _K_dAMP(1 + [ATP]/_K_dATP). This yielded estimates for _K_dAMP and _K_dATP of 51 and 180 μM, respectively, very close to the estimates obtained by direct binding measurements (53 and 175 μM).
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](/articles/view/19874/figure/2)Figure 2
Displacement of AMP from the CBS1-2 construct by ATP. A fixed concentration of [14C]AMP (180 μM) was incubated with the CBS1-2 construct in the presence of increasing concentrations of ATP. Data were fitted to the binding model: bound = AMP/[AMP + _K_dAMP(1 + [ATP]/_K_dATP)].
The CBS3-4 fusion protein bound a single molecule of AMP with a _K_d of 104 μM. We also created a single point mutation in CBS3-4 that causes WPWS when present in intact γ2, i.e., R531G. The mutant still bound AMP, but the _K_d increased 15-fold (Figure 1c and Table 1). The WT also bound ATP with a _K_d fourfold higher than that for AMP, while the _K_d for ATP was increased fivefold relative to the WT in the R531G mutant (Figure 1d and Table 1).
The four tandem CBS domains from AMPK-γ2 bind two molecules of AMP or ATP with positive cooperativity. Since the CBS1-2 and the CBS3-4 fusion proteins from γ2 each bound one molecule of AMP or ATP, we expected that the CBS1-4 construct would bind two, which was indeed the case. The best fits were obtained using a Hill plot model with two identical, interacting sites: bound = 2 × [AMP]h/(B_0.5_h + [AMP]h), where h is the Hill coefficient and _B_0.5 is the concentration giving half-maximal binding (Figure 3a and Table 1). For the WT γ2 construct, this yielded a _B_0.5 for AMP of 61 μM. The Hill coefficient was close to 2, indicating that the two sites bind AMP with strong positive cooperativity, i.e., that once AMP is bound at the first site the affinity at the second site increases so that it fills almost immediately. Similar results were obtained for the binding of ATP (Figure 3b and Table 1), which yielded a _B_0.5 more than fourfold higher than that for AMP, consistent with the finding that both CBS1-2 and CBS3-4 bound ATP with lower affinity than AMP. All WPWS mutants still bound two molecules of AMP, but the B_0.5 values were markedly increased (Figure 3a and Table 1). For L_ins, R302Q, H383R, T400N, and R531G, the increases in _B_0.5 were 2.1-fold, 2.6-fold, 12-fold, 22-fold, and 46-fold, respectively. All mutants also bound two molecules of ATP, but with changes in B_0.5 and/or Hill coefficient (Figure 3b and Table 1), except for the L_ins mutant, which was not significantly different from the WT.
(a and b) Binding of AMP (a) and ATP (b) to the GST–CBS1-4 fusion protein from γ2 and WPWS mutants. (c and d) Binding of AMP (c) and ATP (d) by the GST–CBS1-4 fusion proteins from γ1, γ2, and γ3. The methodology was as for Figure 1, except that data were fitted to a two-site Hill plot model: bound = 2 × [nucleotide]h/(B_0.5_h + [nucleotide]h).
Binding of AMP and ATP to the four tandem CBS domains from AMPK-γ1 and -γ3. We also made CBS1-4 fusion proteins from the γ1 and γ3 isoforms. These also bound two molecules of AMP and ATP with positive cooperativity, but with B0.5 values that were significantly lower (γ1) or higher (γ3) than for γ2 (Table 1, Figure 3, c and d). For the constructs from γ1, γ2, and γ3, the B0.5 values for AMP were 20, 53, and 125 μm, respectively.
Effects of WPWS mutations on activation of AMPK heterotrimers by AMP. While the results in the previous section showed that isolated CBS domain pairs bind AMP and ATP, they did not conclusively prove that these domains form the regulatory nucleotide binding sites in the heterotrimeric AMPK complex. To address this, we expressed recombinant α1β1γ2 heterotrimers in WT and mutated forms in CCL13 cells, purified them by immunoprecipitation via myc epitope tags on the α subunit, and assayed at various AMP concentrations. CCL13 cells (a human cell line now thought to be a variant of HeLa cells) were used because they have a high transfection efficiency and a low endogenous AMPK activity. Figure 4a shows that, for the L_ins_, R302Q, H383R, T400N, and R531G mutations, the _A_0.5 values (concentration causing half-maximal activation) for AMP increased in the same order in which these mutations increased the B_0.5 for binding of AMP to the isolated CBS1-4 constructs, i.e., WT (7 ± 3 μM) ≈ L_ins (9 ± 4 μM) < R302Q (23 ± 6 μM) < H383R (51 ± 40 μM) < T400N (95 ± 32 μM) < R531G (>100 μM). The _A_0.5 values for the heterotrimers were all around tenfold lower than the _B_0.5 values for the isolated CBS1-4 constructs, indicating that the presence of the α and/or β subunits increased the affinity for AMP. It was not possible to conduct assays above 100 μM AMP, because at these concentrations the nucleotide inhibits the activity by competing with ATP at the kinase domain on the α subunit. For this reason, the estimates of _A_0.5 for the H383R and T400N mutants are less accurate than the others, while no value could be obtained for the R531G mutant. The latter appeared to have a slightly elevated basal activity in the absence of AMP and was actually inhibited by addition of AMP. With some mutants, the maximal activation also appeared to be affected. For example, the R302Q mutant was only stimulated threefold by AMP, whereas the WT was stimulated sixfold.
(a) Activation of recombinant α1β1γ2 heterotrimers, with or without WPWS mutations, by AMP. (b) Activation of recombinant α1β1γ1, α1β1γ2, and α1β1γ3 heterotrimers by AMP. (c) Activation of α1β1γ2 heterotrimers, with or without WPWS mutations, by slow versus rapid lysis. Plasmids expressing _myc_-tagged α1 and β1 plus one of the subunits γ1, γ2 (with or without WPWS mutations), and γ3 were expressed in CCL13 cells, and the recombinant complexes were immunoprecipitated using anti-myc antibodies. AMPK activity was then determined at various concentrations of AMP. In a and b, the cells were harvested by slow lysis to elicit maximal phosphorylation (32); in c, the cells were harvested by rapid or slow lysis and the assays were conducted at 200 μM AMP. Results are means ± SE for duplicate immunoprecipitations.
Effects of γ isoform on activation of AMPK heterotrimers by AMP. Figure 4b shows that when the activations of the WT α1β1γ1, α1β1γ2, and α1β1γ3 heterotrimers were compared using the same methodology, they differed in the degree of stimulation by AMP rather than in the _A_0.5 for AMP. The α1β1γ1, α1β1γ2, and α1β1γ3 heterotrimers were stimulated 3.3-, 7.0-, and 1.5-fold by AMP, but the _A_0.5 values were similar (13 ± 2, 12 ± 3, and 2 ± 2 μM, respectively). The value of _A_0.5 for α1β1γ3 is approximate, because of the very small degree of stimulation by AMP with that isoform.
Mutations in γ2 do not cause constitutive activation of AMPK. It has previously been claimed that mutations that are associated with WPWS cause constitutive activation of AMPK (12, 36). To address this, we examined the activation of expressed AMPK in CCL13 cells by slow lysis as opposed to rapid lysis. Slow lysis involves harvesting the cells by scraping them off and centrifuging them prior to resuspension in homogenization medium and activates AMPK by a combination of mechanical stress, hypoxia, and/or glucose deprivation. Rapid lysis involves pouring off the medium and lysing the cells in situ on the culture dish using ice-cold lysis buffer, and this better preserves the physiological phosphorylation status of AMPK. Figure 4c shows that the stress of slow lysis significantly activated the α1β1γ1, α1β1γ2, and α1β1γ3 heterotrimers, although the degree of activation was lowest with the α1β1γ3 complex. All of the γ2 mutants were also activated, although the degree of activation of the R302Q, H383R, T400N, and R531G mutants was significantly lower than that of the WT and the L_ins_ mutant. There was no evidence for constitutive activation of any of the mutants when the cells were harvested by rapid lysis.
The CBS domains from IMPDH bind ATP, and this is impaired by a mutation that causes retinitis pigmentosa. To examine whether binding of adenine nucleotides is a more general function for CBS domains, we also studied IMPDH. Retinitis pigmentosa can be caused by an R224P mutation in the second CBS domain of the IMPDH1 isoform (4). However, IMPDH2 is 84% identical in amino acid sequence with IMPDH1, and the arginine mutated in retinitis pigmentosa is conserved. Because cDNA was more readily available for IMPDH2, we cloned DNA encoding the single pair of CBS domains from that isoform, created an R224P mutation, and expressed the WT and the mutant as polyhistidine-tagged proteins in E. coli. The WT fusion protein bound one molecule of ATP with a _K_d of 54 μM, while in the mutant the _K_d increased more than eightfold (Figure 5a and Table 1). Using displacement of bound ATP as the assay, the WT protein also bound AMP or GMP, but only at supraphysiological concentrations (_K_d = 440 and 5,760 μM, respectively).
(a and b) Binding of ATP by GST fusions of the isolated CBS domain pair (residues 112–232) (a) and full-length IMPDH2 (residues 1–514) (b). (c) Activity of full-length IMPDH2 as a function of ATP concentration. Results were obtained for both the WT sequence and an R224P mutation. Data in a and b were fitted to a single-site binding model as for Figure 1. Data in c were fitted to the model: activity = basal + {[(stimulation × basal) – basal] × [ATP]h}/(A0.5_h_ + [ATP]h).
We also cloned and expressed full-length IMPDH2 and examined the binding of ATP (Figure 5b). Interestingly, this protein (which is a tetramer) bound ATP with a _B_0.5 of 0.76 mM, 14-fold higher than the _K_d for the isolated CBS domains, and much closer to the physiological range of ATP concentrations. An ultraviolet absorption spectrum of the purified protein showed that it did not contain any endogenous bound nucleotide. The binding of ATP to full-length IMPDH2, but not to the isolated CBS domains, also displayed positive cooperativity, with a Hill coefficient of 1.7. ATP binding by the full-length tetramer was also drastically affected by the R224P mutation, with a more than 80-fold increase in _B_0.5 (Figure 5b and Table 1).
ATP is an allosteric activator of IMPDH. Since we could not find any published reports describing effects of ATP on IMPDH activity, we assayed the recombinant enzyme in the presence and absence of the nucleotide. Figure 5c shows that ATP stimulated IMPDH activity more than fourfold with a half-maximal effect at 0.44 ± 0.05 mM and a Hill coefficient of 1.3 ± 0.1. This is close to the _B_0.5 and Hill coefficient obtained for ATP binding to the tetramer. The effect of ATP was on _V_max, rather than on the _K_m for the substrates IMP or NAD+ (not shown). Even at the highest concentration used (2 mM), ATP had no effect on the R224P mutant version of the full-length tetramer (Figure 5c). This is consistent with the insignificant level of binding of ATP to the tetramer at this concentration (Figure 5b).
The CBS domain pair from the chloride channel CLC2 binds ATP, and this is severely affected by pathogenic mutations. We also cloned and expressed DNA encoding the CBS domain pair from the chloride channel CLC2. Figure 6 shows that this construct bound one molecule of ATP with a _K_d of 1.06 ± 0.08 mM, while G715E and G826D mutations were associated with ten- and 14-fold increases in _K_d, respectively.
Binding of ATP by a GST fusion of the isolated CBS domain pair (residues 582–840) from CLC2, and binding of ATP by G715E and G826D mutations. Data were fitted to a single-site binding model as for Figure 1.
The CBS domain pair from cystathionine β-synthase binds S-adenosyl methionine, and this is affected by a homocystinuria mutation. Finally, we cloned and expressed DNA encoding the C-terminal CBS domain pair from the enzyme cystathionine β-synthase. This yielded a preparation that was homogeneous by SDS-PAGE and predominantly migrated as a monomer on native gel electrophoresis (not shown). This domain pair bound one molecule of _S_-adenosyl methionine (SAM) with a _K_d of 34 ± 2 μM, while the D444N mutation that causes homocystinuria (2) increased the _K_d for SAM 15-fold to 510 ± 70 μM (Figure 7).
Binding of SAM by a GST fusion of the isolated CBS domain pair (residues 416–551) from cystathionine β-synthase, and binding of SAM by a D444N mutation. Data were fitted to a single-site binding model as for Figure 1.