Overall, our results show that tandem pairs of CBS domains form allosteric binding sites for adenosine derivatives, i.e., AMP and ATP in the case of AMPK, ATP in the cases of IMPDH and CLC2, and SAM in the case of cystathionine β-synthase. Moreover, they show that the many mutations in CBS domains that cause human hereditary diseases invariably impair the binding of these regulatory adenosine derivatives. To our knowledge, our findings represent not only the first direct evidence as to the function of CBS domains, but also the first description of the biochemical defect in several human hereditary diseases where mutations occur in these domains.
Our results strongly support the idea that the four tandem pairs of CBS domains in the γ subunits of AMPK provide two allosteric binding sites for AMP and ATP in the heterotrimeric complex, one or both of which are therefore targets for development of drugs aimed at obesity and type 2 diabetes. The evidence in favor of this may be summarized as follows: (a) While the N-terminal and C-terminal pairs of CBS domains from the γ2 subunit both bound one molecule of AMP when expressed in and purified from bacteria, the construct containing all four domains bound two molecules of AMP. (b) Mutations that caused an increase in the B0.5
for binding of AMP to the CBS domains of γ2 also caused an increase in the A0.5
for activation of recombinant α1β1γ2 complexes, with the same order of potency, i.e., B0.5
for WT ≈ Lins
< R302Q < H383R < T400N < R531G. (c) ATP also bound to the bacterially expressed CBS domains in a mutually exclusive manner with AMP, albeit with lower affinity. This is consistent with the fact that high concentrations of ATP inhibit allosteric activation of AMPK in a manner that appears to be competitive with AMP (35
). Although mutually exclusive binding to the bacterially expressed CBS domains does not prove that AMP and ATP bind to the same site, this is the simplest explanation that is also consistent with our findings that the γ2 mutations reduced the affinity for ATP in the same order in which they reduced the affinity for AMP.
Our studies on AMP activation of heterotrimeric AMPK complexes are in good agreement with those of Daniel and Carling (37
), who found that mutations associated with WPWS exhibited defective activation by AMP, with the effect on A0.5
increasing in the order WT ≈ Lins
≈ R302Q < H383R (they did not study the T400N mutant). Like we found, Daniel and Carling found that the maximal stimulation by AMP was reduced by the R302Q mutation, while with the R531G mutant the stimulation by AMP was abolished and the basal activity measured in the absence of AMP was slightly higher. They suggested that the latter might be because the inhibitory effect of ATP might also be reduced with this mutant. This is supported by our findings that the affinity of the R531G mutant for ATP, when expressed in the context of either CBS3-4 or CBS1-4 (Table ), was greatly reduced.
Neither our results nor those of Daniel and Carling (37
) support two previous claims (12
) that WPWS mutations make AMPK complexes constitutively active. When the cells were harvested by rapid lysis (which much better preserves the physiological phosphorylation state of AMPK), none of the mutants exhibited greater activity than the WT (Figure c). When the cells were harvested by the slow-lysis procedure (which causes activation of AMPK due to hypoxia, glucose deprivation, and/or mechanical stress), the mutants that exhibited defective activation by AMP in vitro (R302Q, H383R, T400N, and R531G) were all activated, although to a lower extent than the WT. Of the two previous claims that the mutations caused constitutive activation of AMPK, both relied on indirect approaches. One involved making a mutation equivalent to R302Q in the γ1 rather than the γ2 isoform (36
), although naturally occurring mutations in γ1 have not been reported. The other involved making mutations equivalent to T400N and N488I in the yeast γ subunit homologue, Snf4, and analyzing two-hybrid interactions with Snf1 (the α homologue) as a surrogate measure of kinase activity (12
). In fact, while both mutations did appear to cause small (twofold) increases in the Snf1/Snf4 interaction under basal conditions, removal of glucose from the medium (known to activate the SNF1 complex [ref. 38
]) appeared to cause further large increases in interaction with both of the mutants as well as with the WT. The use of the term “constitutively active” to describe these Snf4 mutants (12
) is therefore misleading. Our results and those of Daniel and Carling (37
) suggest that while the R531G mutant has a slightly elevated basal activity (due perhaps to reduced binding of the inhibitor, ATP), the major effect of the mutations is to reduce
activation of AMPK in response to stress.
Our results with IMPDH2 and CLC2 suggest that sensing of cellular energy status by binding of adenine nucleotides may be a general function of CBS domain pairs, rather than being a function restricted to AMPK. The CBS domain pairs from IMPDH2 also bound AMP and GMP in vitro in addition to ATP, but only ATP bound at physiologically relevant concentrations. IMPDHs catalyze the first step in purine nucleotide biosynthesis that is committed to synthesis of GMP rather than AMP. They are subject to feedback inhibition by GMP, but GMP did not bind with high affinity to the CBS domains. Since inhibition by GMP is competitive with the substrate, IMP (39
), it is more likely that feedback inhibition is due to binding of GMP to the catalytic site. It is interesting that, while ATP bound to the isolated CBS domain pair from IMPDH2 with a Kd
in the low micromolar range (54 μM) with no evidence for interaction between sites, it bound to full-length enzyme with positive cooperativity and a B0.5
(770 μM) that was 14-fold higher and much closer to the physiological range of ATP concentrations. The crystal structures of mammalian and bacterial IMPDH show that the enzyme is a tetramer with the CBS domain pairs on the outside, with subunit contacts being made entirely by the catalytic domains (34
). Our results suggest that the CBS domain pairs in the tetramer are constrained in a conformation that has a lower affinity for ATP than the isolated domains have, but that binding of ATP to the first site causes a conformational change that is transmitted across the subunit interface to increase the affinity of the remaining CBS domain pairs.
We were unable to find any previous reports that IMPDH was activated by ATP, but our results now show, for the first time to our knowledge, that the nucleotide increases the Vmax
of the enzyme more than fourfold. The reasonably close correspondence between the B0.5
for ATP binding (770 μM) and the A0.5
for ATP activation (440 μM) of the tetramer, and the finding that the R224P mutation in the second CBS domain abolishes both binding of ATP and activation by ATP, provide strong evidence that the allosteric activation is due to binding of the nucleotide to the CBS domains. We propose that this mechanism ensures that the synthesis of guanine nucleotides only occurs when the supply of ATP is sufficient, a mechanism that accompanies feedback inhibition by GMP. This is analogous to the regulation of the key enzyme of pyrimidine nucleotide synthesis, aspartate transcarbamylase, in bacteria, which is inhibited by the end product CTP while being activated by ATP (41
Our results also suggest that the natural ligand for the CBS domains of CLC chloride channels is ATP, and that a mutation in CLC2 (G715E) associated with idiopathic generalized epilepsy (6
), and a mutation (G826D) equivalent to one in CLC1 that causes congenital myotonia (5
), both lead to a severe defect in ATP binding. While our work was in progress, Vanoye and George (42
) reported, using patch clamp analysis, that human CLC4 channels only supported a chloride current when incubated on the cytoplasmic side with ATP or a nonhydrolyzable analogue. The CBS domain pairs of the CLC family are predicted to lie on the cytoplasmic side of the membrane. Our results and those of Vanoye and George suggest that binding of ATP to the CBS domains of the CLC chloride channels is necessary before the channels will open.
We therefore propose that, in most cases, tandem pairs of CBS domains form sensors of cellular energy status that act by binding AMP and/or ATP. An exception to this appears to be cystathionine β-synthase itself, which catalyzes the first step in cysteine synthesis and is allosterically activated by SAM. Its substrate, homocysteine, is an intermediate in the “activated methyl cycle” in which SAM (an important donor of methyl groups during biosynthesis) is regenerated from S
-adenosyl homocysteine. A low activity of cystathionine β-synthase would promote recycling of homocysteine into SAM, whereas activation of the enzyme by high concentrations of SAM would favor removal of homocysteine from the cycle and its conversion to cysteine instead. Intriguingly, point mutations in the CBS domains of cystathionine β-synthase, or premature termination or proteolysis that removes them, result in enzyme that no longer responds to SAM (2
). Kraus and coworkers (33
) provided evidence that the CBS domains form an autoinhibitory domain whose effect is relieved by SAM binding, which is consistent with the idea that the CBS domains might represent the binding site for SAM, although this had not been directly demonstrated. Our present results now provide strong support for this hypothesis, since the isolated CBS domains bound one molecule of SAM with a Kd
of 34 μM, while the D444N mutation increased this to approximately 500 μM (Figure ). These results are consistent with previous estimates of SAM binding to the full-length enzyme of around 15 μM (33
), and with results showing that WT cystathionine β-synthase was activated by SAM with a half-maximal effect at 7 μM, while in the D444N mutant this was increased to 460 μM (45
). The pathogenic effect of the D444N mutation might also be due in part to a reduced expression of the protein (45
In the case of cystathionine β-synthase, the CBS domains appear to have been adapted to bind not an adenine nucleotide, but a related adenosine-containing compound, SAM. In fact the second CBS domain in cystathionine β-synthase is rather poorly conserved (ref. 1
; see also the PFAM database, entry no. PF00571, ref. 46
). In addition, a truncation that removes the N-terminal 70 residues of cystathionine β-synthase, which contains the heme-binding region, results in enzyme that retains 20% of WT activity but is no longer activated by SAM (47
). One possibility is that the CBS domains of cystathionine β-synthase bind the adenosyl moiety of SAM, whereas the N-terminal heme domain is involved in binding of the methionine moiety, which is not present in ligands that bind to other CBS domain pairs.
Although our present results provide strong support for the idea that the CBS domains of the γ subunits of AMPK provide the allosteric binding sites for AMP and ATP, a number of puzzling findings remain to be addressed in future studies. Firstly, the A0.5
values for activation of WT and mutant versions of the α1β1γ2 complex by AMP were generally around tenfold lower than the B0.5
values for binding of AMP to the equivalent CBS1-4 constructs. Secondly, the B0.5
for binding of AMP to the WT CBS1-4 constructs was different for the three γ subunit isoforms (γ1, 20 μM; γ2, 53 μM; γ3, 125 μM), although the recombinant α1β1γ1, α1β1γ2, and α1β1γ3 complexes were all activated by similar, lower concentrations of AMP (A0.5
= 2–13 μM) and differed instead in their degree of stimulation by AMP (Figure b), in agreement with previous results obtained with native complexes prepared by immunoprecipitation (28
). Another unexpected finding was that the CBS domains of γ2 contain two binding sites for both AMP and ATP. There is an interesting parallel here with the regulatory subunits of cAMP-dependent protein kinase, which contain two tandem binding sites for cAMP, although these are not related to CBS domains. A current model for activation of cAMP-dependent protein kinase is that cAMP binds initially to the C-terminal site (site B), and only then does site A become accessible, although it is binding to site A that causes activation (48
). Further work is required to determine whether a similar mechanism operates in the case of AMPK. The finding that mutations in CBS1 (R302Q), CBS2 (H383R and T400N), and CBS4 (R531G) all affect AMP activation (Figure a) suggests that AMP must bind to both sites for activation to occur. However, a sigmoidal activation of AMPK by AMP (which might be expected if occupancy of both binding sites were necessary for activation) has not been reported to our knowledge.
Another puzzling finding is that, although the Saccharomyces cerevisiae
homologues of the AMPK γ subunits (Snf4p) and cystathionine β-synthase both contain CBS domains in the same position as their human counterparts, they have not been found to be activated by AMP (38
) or SAM (49
Finally, one interesting feature of the pathogenic mutations in CBS domains is that they tend to occur in equivalent positions. Thus, the R302Q, H383R, and R531G mutations in CBS1, CBS2, and CBS4 of γ2 all align (plus or minus one residue) with the D444N mutation in cystathionine β-synthase and with the R200Q mutation in CBS1 of the γ3 isoform of pig AMPK (13
). Similarly, the T400N mutation in CBS2 of γ2 aligns (plus or minus one residue) with the R224P mutation in CBS2 of IMPDH1. These “hot spots” for pathogenic mutations are likely to be directly involved in binding of the adenosine-containing ligand. A model for the N-terminal domain pair in the γ2 subunit of AMPK, based on the atomic coordinates of a bacterial IMPDH (34
), is shown in Figure . Two four-stranded β sheets form a deep hydrophobic cleft between the two domains that is of suitable dimensions to accommodate an adenosine moiety. Many of the residues that are mutated in disease states have basic, positively charged side chains (e.g., Arg302 and His383 in γ2, and Arg224 in IMPDH2, which aligns with His401 in γ2) and are predicted to lie around the mouth of this cleft (Figure ). In the CBS domain pairs that bind AMP and/or ATP, these residues form a positively charged patch at the mouth of the cleft, which is likely to bind the α phosphate moiety of adenine nucleotides. Other residues that are mutated (Thr400 in γ2, and Gly828 in CLC2, which aligns with Tyr397 in γ2) are predicted to lie within the cleft itself, where they may form interactions with the ribose or adenine moieties. Another notable feature is that the basic residues that form the basic, positively charged patch at the mouth of the cleft in γ2 are either uncharged or acidic in cystathionine β-synthase. The D444N mutation in the latter protein would neutralize a negative charge that occurs in the equivalent position to Arg302 in γ2. Intriguingly, the ligand for cystathionine β-synthase, i.e., SAM, has a positively charged sulfur atom in approximately the same position as the negatively charged phosphate of AMP, and it is likely that Asp444 forms an electrostatic interaction with this sulfur atom.
Figure 8 Model of the N-terminal pair of CBS domains (CBS1 and CBS2) from the γ2 subunit of AMPK. The picture is a view of a molecular-surface representation made using the program GRASP (Department of Biochemistry, Columbia University, New York, New York, (more ...)