Formation of the Productive ATP-Mg 2+ -bound Dimer of GlcV, an ABC-ATPase from Sulfolobus solfataricus

Abstract

The ABC-ATPase GlcV from Sulfolobus solfataricus energizes an ABC transporter mediating glucose uptake. In ABC transporters, two ABC-ATPases are believed to form a head-to-tail dimer, with both monomers contributing conserved residues to each of the two productive active sites. In contrast, isolated GlcV, although active, behaves apparently as a monomer in the presence of ATP-Mg 2þ , AMPPNP-Mg 2þ or ATP alone. To resolve the oligomeric state of the active form of GlcV, we analysed the effects of changing the putative catalytic base, residue E166, into glutamine or alanine. Both mutants are, to different extents, defective in ATP hydrolysis, and gel-filtration experiments revealed their dimerization in the presence of ATP-Mg 2þ . Mutant E166Q forms dimers also in the presence of ATP alone, without Mg 2þ , whereas dimerization of mutant E166A requires both ATP and Mg 2þ . These results confirm earlier reports for other ABCATPases, but for the first time suggest the occurrence of a fast equilibrium between ATP-bound monomers and ATP-bound dimers. We further mutated two highly conserved residues of the ABC signature motif, S142 and G144, into alanine. The G144A mutant is completely inactive and fails to dimerize, indicating an essential role of this residue in stabilizing the productive dimeric state. Mutant S142A retained considerable activity, and was able to dimerize, thus implying that the interaction of the serine with ATP is not essential for dimerization and catalysis. Furthermore, although the E166A and G144A mutants each alone are inactive, they produce an active heterodimer, showing that disruption of one active site can be tolerated. Our data suggest that ABC-ATPases with partially degenerated catalytic machineries, as they occur in vivo, can still form productive dimers to drive transport.

Thermodynamics of the ATPase Cycle of GlcV, the Nucleotide-Binding Domain of the Glucose ABC Transporter of Sulfolobus solfataricus

“…As observed in the gel filtration experiments, the E166A mutant stably dimerizes in an ATP-dependent manner both in the presence and in the absence of Mg 2+ ( Figure 1B). On the other hand, the G144A mutant showed a translational diffusion coefficient of 644 × 10 -9 cm 2 /s and remained unaffected by the ATP concentration, confirming the previous findings that this mutant remains monomeric in the presence of ATP (23). The maximal (∼645 × 10 -9 cm 2 /s) and minimal (∼570 × 10 -9 cm 2 /s) translational diffusion coefficients corresponded to globular proteins of 54 and 73 kDa, respectively.…”

“…The G144A and E166A mutants, which carry mutations in the C-loop and in the residue directly after the aspartate of the Walker B binding motif, have strongly reduced ATPase activity (23). In the presence of ATP, the E166A mutant of GlcV exhibited ATP-dependent dimerization as shown by size-exclusion chromatography, whereas wt GlcV and the G144A mutant eluted as monomers (23). This dimerization, however, is observed only when ATP is present in the elution buffer, suggesting that the E166A dimer is in a dynamic equilibrium with the monomer and readily dissociates on the size-exclusion column.…”

“…The ATPase activity of wild-type GlcV is too high to allow the detection of a dimer in solution. Mutation of the conserved glutamate downstream of the Walker B motif of GlcV (E166Q) resulted in a strongly reduced ATPase activity, and this allowed demonstration of ATP-dependent dimerization of GlcV (23). Substitution of the glutamate for alanine resulted in a GlcV mutant that is inactive for ATP hydrolysis and that required both ATP and Mg 2+ for dimer formation.…”

Positive co-operative activity and dimerization of the isolated ABC ATPase domain of HlyB from Escherichia coli

“…Attempts to overexpress either NBD1 or NBD2 of human Mdr1 in soluble form in our laboratory have been similarly unsuccessful (H. Benabdelhak and M. A. Blight, unpublished work). Several recent successful structural studies of ABC (NBD) domains have been obtained with proteins derived from thermophiles, and this may avoid this insolubility problem [10,38,39]. Unfortunately, however, this approach is offset by the lack of any knowledge so far of the mechanism of action of such thermophile proteins in vivo.…”

“…However, while the stoichiometry of the in vivo or reconstituted histidine and maltose uptake systems indicates the presence of two molecules of the separately encoded HisP or MalK ABC ATPases [5,6], direct evidence for stable dimerization of individual ABC NBDs (nucleotide-binding domains) in purified form, even in the presence of nucleotides, has been difficult, if not impossible, to obtain [7][8][9][10][11]. On the other hand, OpuAA, required for uptake of glycine betaine in Bacillus subtilis, does form dimers, independently of ATP [11].…”