Mutational analyses of the bacterial cassettes HisP (14
) and KpsT (18
) and of mouse Mdr3 (19
) indicate that a highly conserved glutamic acid residue, found directly adjacent to the Walker B aspartic acid residue in the sequence RX6 – 8
P (), is critical for ATPase and transport activity. This glutamic acid residue is in position to activate a nearby water in the HisP structure and was thus predicted to serve as the catalytic carboxylate (7
). To further investigate the role of this glutamic acid residue, we generated either glutamate to glutamine or alanine mutations in MJ0796 and MJ1267, two well characterized ABC transporter nucleotide binding cassettes (9
ATPase activity was assayed using a coupled ATPase assay (17
). Wild type MJ0796 (1 μ
M) exhibited a Vmax
of 0.2 s−1
with a Km
of 50 μ
M (). The Hanes-Woolf plot (, inset
) presents a shape diagnostic of positively cooperative ATP binding/hydrolysis consistent with the determined Hill coefficient of 1.7. Positive, two-site cooperativity was previously observed for wild type bacterial maltose (22
) and histidine (23
) transporters. By contrast, the E171Q mutant of MJ0796 had undetectable ATPase activity (). The equivalent mutation in the MJ1267 cassette (E179Q) also eliminated its ATPase activity (data not shown). These results show that the carboxylate functional group of the highly conserved glutamate residue at the C terminus of the Walker B sequence () is required for ATP hydrolysis.
Based on the Rad50 model for the transport cycle, the hydrolysis-deficient glutamate to glutamine mutants of ABC transporter nucleotide binding cassettes would be expected to form a similar ATP-bound homodimer provided their nucleotide binding ability is unaltered by the mutation. To assay the ability of mutant cassettes to form stable, nucleotide-dependent dimers, we performed experiments wherein protein samples were mixed with ADP or ATP and then resolved on a size exclusion column. The data () show that both wild type and E171Q mutant MJ0796 migrated as monomers in the absence of ATP. However, the E171Q mutant migrated largely as a homodimer in the presence of ATP (). Wild type MJ0796 migrated as a monomer regardless of the presence of ATP (). ADP did not support the formation of a stable dimer of either wild type () or mutant () MJ0796. Moreover the addition of ADP inhibited ATP-dependent dimer formation in the E171Q mutant (data not shown). The fact that the mutant ATP-containing dimer was stable during elution in the absence of nucleotide in the mobile phase indicates that the nucleotide sandwich dimers only slowly dissociate in the absence of ATP hydrolysis. Consistent with our findings, previous studies of wild type HisP (24
) and MalK (25
) exhibit no more than a small degree of dimerization in the presence of nucleotide.
Analytical gel filtration assays of nucleotide-dependent cassette dimerization
The dependence of the dimerization of the E171Q mutant of MJ0796 on ATP concentration is shown in the analytical gel filtration data in . The midpoint of the titration was between 50 and 100 μM, consistent with the Km for ATP hydrolysis observed for wild type MJ0796 (). Analogous results demonstrating the ATP-dependent dimerization of the E179Q mutant of the MJ1267 nucleotide binding cassette are shown in . The midpoint of the titration occurred at a slightly higher ATP concentration for this protein. Like MJ0796, wild type MJ1267 did not form stable dimers in the presence of ATP (data not shown).
To determine the energetics of the ATP-dependent homodimerization of MJ0796-E171Q observed by analytical gel filtration, samples were analyzed at a protein concentration of 56 μ
M by equilibrium analytical ultracentrifugation (data summarized in ). In the absence of ATP, a KD
of 208 μ
M was calculated for monomer-homodimer equilibrium, indicating that the protein was present primarily as a monomer under these conditions, consistent with the gel filtration results (). Again ADP did not support dimerization in the equilibrium analytical ultracentrifugation analysis. By contrast, the addition of 2 mM ATP resulted in a reduction of the KD
to 70 nM, indicating that the protein was present primarily as a dimer under these conditions. The steep dependence of the KD
on ATP concentration is expected if dimerization is coupled to positively cooperative ATP binding in the symmetric sandwich dimer (27
The non-hydrolyzable ATP analogues ATPγS and AMP-PNP failed to promote cassette dimerization in gel filtration experiments on wild type MJ0796 and MJ1267 (data not shown) and only poorly promoted dimerization of the mutant cassettes (). These results suggest that these analogues, while useful in numerous other applications, are not always accurate mimetics of their natural counterparts. The subtle electrostatic and steric differences between conventional nucleotides and the non-hydrolyzable analogs apparently prevent stable cassette dimerization. This observation likely explains the difficulty experienced in isolating wild type cassette dimers in the presence of non-hydrolyzable ATP analogues.
Effect of non-hydrolyzable nucleotide analogues and cations on dimerization of MJ0796-E171Q and MJ1267-E179Q
The addition of Mg2+
or the substitution of K+
during analytical gel filtration and ultracentrifugation experiments inhibited the formation of the MJ0796-E171Q dimer ( and ). Addition of 10 mM MgCl2
lowered the dimer level by ~25% in the presence of 10 mM ATP. Likewise a decrease in dimerization was seen when KCl was substituted for NaCl ( and ). In KCl the KD
for dimerization increased from 20 to 600 nM. Interestingly mutating the catalytic glutamate in MJ1267 to alanine (E179A) diminished ATP-dependent dimerization to 10% of the level achieved with the E179Q mutant (). The ability of these modifications of the ionic environment to alter the energetics of dimer formation reinforces the idea that carefully balanced electrostatic effects play a critical role in mediating the ATP-dependent dimerization of ABC transporter nucleotide binding cassettes. Upon ATP hydrolysis, additional alterations in the electrostatics of the interface due to deprotonations and/or product release could effectively destabilize the sandwich dimer (). The structure of the stable ATP sandwich dimer of MJ0796-E171Q has been solved by x-ray crystallography and details the specific nature of these interactions.2
A model for the reaction cycle of ABC transporters based on ATP-dependent dimerization of ATP-binding cassettes
Interestingly in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel (28
), only the C-terminal cassette contains the catalytic glutamate residue. A recent study of the non-equivalency of CFTR cassettes demonstrated that nucleotide binds stably and dissociates slowly from the N-terminal cassette, while the C-terminal cassette rapidly hydrolyzes nucleotide (26
). Thus, based on the data presented here, it is possible that the N-terminal cassette of CFTR, which contains a serine rather than glutamate at the end of the Walker B sequence, forms associations that stabilize the protein in a substate that has channel activity.
Conformational changes coupled to hydrolysis of the bound ATP also likely play a role in driving the cassettes apart (). Comparison of ADP-bound conformations of the MJ0796 and MJ1276 cassettes with the ATP-bound form of the HisP cassette suggests that ATP binding produces a significant rotation of an α
-helical subdomain in the cassettes accompanied by rearrangement of the γ
-phosphate linker segment and rotation of a conserved histidine residue out of the active site (7
). However, this subdomain rotation is unlikely to be the power stroke of the transport process since the mutation of the phylogenetically invariant glutamine residue (the equivalent of Gln-90 in MJ0796 and Gln-89 in MJ1267) mediating the rotation slows but does not abolish transport.3
In this regard, while these subdomain rearrangements are most likely critical for determining the rate of product release, the best candidate for the power stroke of ABC transporters is the ATP-driven formation of the cassette dimer as proposed by Hopfner et al.
) and verified in this study. In this model, in the intact transporter the cassette dimer-monomer transition would be coupled to conformational changes in the TM domains that modulate the affinity and differential exposure of a transport substrate binding site on alternating sides of the membrane barrier.