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A single acidic residue can guide binding site selection but does not govern QacR cationic-drug affinity.

Peters KM, Brooks BE, Schumacher MA, Skurray RA, Brennan RG, Brown MH - PLoS ONE (2011)

Bottom Line: Here, we report structural, biochemical and in vivo effects of substituting these glutamate residues.Structures of QacR(E90Q) and QacR(E120Q) with ethidium or malachite green took similar global conformations that differed significantly from all previously described QacR-drug complexes but still prohibited binding to cognate DNA.Thus, these data strongly suggest that rather than contributing significantly to ligand binding affinity, the role of acidic residues lining the QacR multidrug-binding pocket is primarily to attract and guide cationic drugs to the "best available" positions within the pocket that elicit QacR induction.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Sydney, Sydney, New South Wales, Australia.

ABSTRACT
Structures of the multidrug-binding repressor protein QacR with monovalent and bivalent cationic drugs revealed that the carboxylate side-chains of E90 and E120 were proximal to the positively charged nitrogens of the ligands ethidium, malachite green and rhodamine 6G, and therefore may contribute to drug neutralization and binding affinity. Here, we report structural, biochemical and in vivo effects of substituting these glutamate residues. Unexpectedly, substitutions had little impact on ligand affinity or in vivo induction capabilities. Structures of QacR(E90Q) and QacR(E120Q) with ethidium or malachite green took similar global conformations that differed significantly from all previously described QacR-drug complexes but still prohibited binding to cognate DNA. Strikingly, the QacR(E90Q)-rhodamine 6G complex revealed two mutually exclusive rhodamine 6G binding sites. Despite multiple structural changes, all drug binding was essentially isoenergetic. Thus, these data strongly suggest that rather than contributing significantly to ligand binding affinity, the role of acidic residues lining the QacR multidrug-binding pocket is primarily to attract and guide cationic drugs to the "best available" positions within the pocket that elicit QacR induction.

Show MeSH
Convergent QacR mutant E90Q and wild type QacR drug-binding sites.Superimposition of the structures of the multidrug-binding pockets of wild type QacR-malachite green (MG; light blue ribbon), QacR(E90Q)-ethidium (Et; pink ribbon), and QacR(E90Q)-rhodamine 6G (R6G; yellow ribbon) structures. Helices containing drug-interacting residues are labelled in black.
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pone-0015974-g004: Convergent QacR mutant E90Q and wild type QacR drug-binding sites.Superimposition of the structures of the multidrug-binding pockets of wild type QacR-malachite green (MG; light blue ribbon), QacR(E90Q)-ethidium (Et; pink ribbon), and QacR(E90Q)-rhodamine 6G (R6G; yellow ribbon) structures. Helices containing drug-interacting residues are labelled in black.

Mentions: Additional intriguing dimensions to the recognized promiscuity of QacR-multidrug interactions are highlighted by the significantly altered binding position of Et in the QacR(E90Q)-Et complex and the two distinct R6G binding sites found in the structure of QacR(E90Q)-R6G. Although charge neutralization of Et was ascribed solely to E120 in the wt QacR-drug complex [3], the location of Et in the QacR(E90Q)-Et structure is dramatically altered (Figures 2B and 4). Indeed, 2Fo-Fc composite omit maps revealed strong and full electron density for Et in this newly revealed binding site despite having relatively high average B-factors (Figure S6B, Table S1). In this QacR(E90Q)-Et complex, Et is angled at 120° relative to its wt orientation and shifted toward the R6G sub-pocket, hence overlapping the Et and R6G sub-pockets and taking a position that is comparable to that occupied by MG (Figure 4). From this location Et now interacts with a new complement of residues distinct from that observed in the wt QacR-Et complex, with a number of drug contacts gained and lost. Lost are van der Waals contacts with the side chains of I99 and I100 and aromatic stacking interactions with F162′ and Y103, the latter residue shifting from a position that would have clashed with Et in the QacR(E90Q)-Et structure. Nevertheless, Et gains stacking interactions with W61 and van der Waals contacts with S86, M116 and N154. Further, the drug maintains contacts with the side chains of residues Q96, E120, Y123 and N157, although the nature of these interactions, in some instances, is altered dramatically. Indeed, in the QacR(E90Q)-Et structure, the side chain of E120, despite remaining within 4 Å of the Et phenanthridinium ring system, is repositioned to avoid a clash with the drug, shifting by 3.0 Å so that it is no longer available to charge complement the Et N5 nitrogen atom (E120-N5 distance  = 4.0 Å in the wt QacR-Et complex and 7.0 Å in the QacR(E90Q)-Et complex). This lost interaction is replaced by a cation-π interaction with Y123 which now stacks with Et. Additionally, the side chain of E90Q swings further into the binding pocket to a position comparable to that observed in the QacR(E90Q)-Dq structure (Figures 2A–B). Thus, relocation of Et in the binding pocket of QacR(E90Q)-Et is apparently, although somewhat enigmatically, driven by the QacR mutation E90Q and subsequent reorientation of the side chains of E90Q, Y103 and E120. Despite the loss of interaction with E90 and employing an almost entirely new complement of QacR residues, this new binding site in the QacR(E90Q)-Et complex exhibited binding affinity and induction capabilities for Et essentially the same as wt QacR (Tables 1 and 2).


A single acidic residue can guide binding site selection but does not govern QacR cationic-drug affinity.

Peters KM, Brooks BE, Schumacher MA, Skurray RA, Brennan RG, Brown MH - PLoS ONE (2011)

Convergent QacR mutant E90Q and wild type QacR drug-binding sites.Superimposition of the structures of the multidrug-binding pockets of wild type QacR-malachite green (MG; light blue ribbon), QacR(E90Q)-ethidium (Et; pink ribbon), and QacR(E90Q)-rhodamine 6G (R6G; yellow ribbon) structures. Helices containing drug-interacting residues are labelled in black.
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Related In: Results  -  Collection

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pone-0015974-g004: Convergent QacR mutant E90Q and wild type QacR drug-binding sites.Superimposition of the structures of the multidrug-binding pockets of wild type QacR-malachite green (MG; light blue ribbon), QacR(E90Q)-ethidium (Et; pink ribbon), and QacR(E90Q)-rhodamine 6G (R6G; yellow ribbon) structures. Helices containing drug-interacting residues are labelled in black.
Mentions: Additional intriguing dimensions to the recognized promiscuity of QacR-multidrug interactions are highlighted by the significantly altered binding position of Et in the QacR(E90Q)-Et complex and the two distinct R6G binding sites found in the structure of QacR(E90Q)-R6G. Although charge neutralization of Et was ascribed solely to E120 in the wt QacR-drug complex [3], the location of Et in the QacR(E90Q)-Et structure is dramatically altered (Figures 2B and 4). Indeed, 2Fo-Fc composite omit maps revealed strong and full electron density for Et in this newly revealed binding site despite having relatively high average B-factors (Figure S6B, Table S1). In this QacR(E90Q)-Et complex, Et is angled at 120° relative to its wt orientation and shifted toward the R6G sub-pocket, hence overlapping the Et and R6G sub-pockets and taking a position that is comparable to that occupied by MG (Figure 4). From this location Et now interacts with a new complement of residues distinct from that observed in the wt QacR-Et complex, with a number of drug contacts gained and lost. Lost are van der Waals contacts with the side chains of I99 and I100 and aromatic stacking interactions with F162′ and Y103, the latter residue shifting from a position that would have clashed with Et in the QacR(E90Q)-Et structure. Nevertheless, Et gains stacking interactions with W61 and van der Waals contacts with S86, M116 and N154. Further, the drug maintains contacts with the side chains of residues Q96, E120, Y123 and N157, although the nature of these interactions, in some instances, is altered dramatically. Indeed, in the QacR(E90Q)-Et structure, the side chain of E120, despite remaining within 4 Å of the Et phenanthridinium ring system, is repositioned to avoid a clash with the drug, shifting by 3.0 Å so that it is no longer available to charge complement the Et N5 nitrogen atom (E120-N5 distance  = 4.0 Å in the wt QacR-Et complex and 7.0 Å in the QacR(E90Q)-Et complex). This lost interaction is replaced by a cation-π interaction with Y123 which now stacks with Et. Additionally, the side chain of E90Q swings further into the binding pocket to a position comparable to that observed in the QacR(E90Q)-Dq structure (Figures 2A–B). Thus, relocation of Et in the binding pocket of QacR(E90Q)-Et is apparently, although somewhat enigmatically, driven by the QacR mutation E90Q and subsequent reorientation of the side chains of E90Q, Y103 and E120. Despite the loss of interaction with E90 and employing an almost entirely new complement of QacR residues, this new binding site in the QacR(E90Q)-Et complex exhibited binding affinity and induction capabilities for Et essentially the same as wt QacR (Tables 1 and 2).

Bottom Line: Here, we report structural, biochemical and in vivo effects of substituting these glutamate residues.Structures of QacR(E90Q) and QacR(E120Q) with ethidium or malachite green took similar global conformations that differed significantly from all previously described QacR-drug complexes but still prohibited binding to cognate DNA.Thus, these data strongly suggest that rather than contributing significantly to ligand binding affinity, the role of acidic residues lining the QacR multidrug-binding pocket is primarily to attract and guide cationic drugs to the "best available" positions within the pocket that elicit QacR induction.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Sydney, Sydney, New South Wales, Australia.

ABSTRACT
Structures of the multidrug-binding repressor protein QacR with monovalent and bivalent cationic drugs revealed that the carboxylate side-chains of E90 and E120 were proximal to the positively charged nitrogens of the ligands ethidium, malachite green and rhodamine 6G, and therefore may contribute to drug neutralization and binding affinity. Here, we report structural, biochemical and in vivo effects of substituting these glutamate residues. Unexpectedly, substitutions had little impact on ligand affinity or in vivo induction capabilities. Structures of QacR(E90Q) and QacR(E120Q) with ethidium or malachite green took similar global conformations that differed significantly from all previously described QacR-drug complexes but still prohibited binding to cognate DNA. Strikingly, the QacR(E90Q)-rhodamine 6G complex revealed two mutually exclusive rhodamine 6G binding sites. Despite multiple structural changes, all drug binding was essentially isoenergetic. Thus, these data strongly suggest that rather than contributing significantly to ligand binding affinity, the role of acidic residues lining the QacR multidrug-binding pocket is primarily to attract and guide cationic drugs to the "best available" positions within the pocket that elicit QacR induction.

Show MeSH