Nucleotide-Binding Sites of the Heterodimeric LmrCD ABC-Multidrug Transporter of Lactococcus lactis Are Asymmetric

Abstract

LmrCD is a lactococcal, heterodimeric multidrug transporter, which belongs to the ABC superfamily. It consists of two half-transporters, LmrC and LmrD, that are necessary and sufficient for drug extrusion and ATP hydrolysis. LmrCD is asymmetric in terms of the conservation of the functional motifs of the nucleotide-binding domains (NBDs). Important residues of the nucleotide-binding site of LmrC and the C loop of LmrD are not conserved. To investigate the functional importance of the LmrC and LmrD subunits, the putative catalytic base residue adjacent to the Walker B motif of both NBDs were substituted for the respective carboxamides. Our data demonstrate that Glu587 of LmrD is essential for both drug transport and ATPase activity of the LmrCD heterodimer, whereas mutation of Asp495 of LmrC has a less severe effect on the activity of the complex. Structural and/or functional asymmetry is further demonstrated by differential labeling of both subunits by 8-azido-[α-32P]ATP, which, at 4 °C, occurs predominantly at LmrC, while aluminiumfluoride (AlF x )-induced trapping of the hydrolyzed nucleotide at 30 °C results in an almost exclusive labeling of LmrD. It is concluded that the LmrCD heterodimer contains two structurally and functionally distinct NBDs.

Engineering ATPase Activity in the Isolated ABC Cassette of Human TAP1

“…Only this allows a direct unbiased comparison of the enzymes employed here and explains the relatively low activity for the double mutant. Finally, our model can explain the recent report on the asymmetric behavior of the motor domains of the ABC-transporter LmrCD from L. lactis (50). Due to the nature of the amino acids forming the catalytic dyad (aspartate/glutamine in LmrC and glutamate/histidine in LmrD), ATPase activity should be asymmetric.…”

“…Accordingly, LmrC would display no detectable activity, whereas LmrD should be capable of hydrolyzing ATP. This is exactly reflected in the biochemical and mutational studies of the heterodimeric LmrCD complex (50). However, we like to stress here, that further systems have to be investigated before a generalization can be drawn.…”

“…This situation is reminiscent of the recently described bacterial multidrug tranporter LmrCD from Lactococcus lactis. LmrC shows the above indicated deviations from the canonical sequences and in terms of primary structure would correspond to TAP1, whereas LmrD would correspond to TAP2 (50). Furthermore, the C-loop of TAP2 and the C-terminal ABC cassettes of CFTR and MRP1 do not contain the consensus sequence (LSGGQ).…”

Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

“…Given that AlFx, like vanadate, functions as a transition state analogue for ATP hydrolysis, these results strongly suggest that LmrD is more catalytically active than LmrC. This observation is interesting because a similar catalytic asymmetry occurs in eukaryotic ABC transporters, such as MRP1, CFTR, SUR1, PDR5, and TAP (296,347).…”

“…Interestingly, the two NBDs differ with respect to the conservation of several important residues, including the conserved glutamate following the Walker B motif, which is known to be involved in ATP hydrolysis (347). Mutation of this residue suggested a structural and/or functional asymmetry in the NBDs of LmrCD (296). Residue Glu587 in LmrD proved to be essential for both drug transport and ATPase activity of LmrCD, whereas mutation of the equivalent residue, Asp495, in LmrC has a less severe effect on the activity of the heterodimer.…”