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Structural mechanism of the simultaneous binding of two drugs to a multidrug-binding protein.

ABSTRACT: The structural basis of simultaneous binding of two or more different drugs by any multidrug-binding protein is unknown and also how this can lead to a noncompetitive, uncompetitive or cooperative binding mechanism. Here, we describe the crystal structure of the Staphylococcus aureus multidrug-binding transcription repressor, QacR, bound simultaneously to ethidium (Et) and proflavin (Pf). The structure underscores the plasticity of the multidrug-binding pocket and reveals an alternative, Pf-induced binding mode for Et. To monitor the simultaneous binding of Pf and Et to QacR, as well as to determine the effects on the binding affinity of one drug when the other drug is prebound, a novel application of near-ultraviolet circular dichroism (UVCD) was developed. The UVCD equilibrium-binding studies revealed identical affinities of Pf for QacR in the presence or absence of Et, but significantly diminished affinity of Et for QacR when Pf is prebound, findings that are readily explicable by their structures. The principles for simultaneous binding of two different drugs discerned here are likely employed by the multidrug efflux transporters.

SUBMITTER: Schumacher MA

PROVIDER: S-EPMC514915 | biostudies-literature | 2004 Aug

REPOSITORIES: biostudies-literature

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Structural mechanism of the simultaneous binding of two drugs to a multidrug-binding protein.

Schumacher Maria A MA Miller Marshall C MC Brennan Richard G RG

The EMBO journal 20040715 15

The structural basis of simultaneous binding of two or more different drugs by any multidrug-binding protein is unknown and also how this can lead to a noncompetitive, uncompetitive or cooperative binding mechanism. Here, we describe the crystal structure of the Staphylococcus aureus multidrug-binding transcription repressor, QacR, bound simultaneously to ethidium (Et) and proflavin (Pf). The structure underscores the plasticity of the multidrug-binding pocket and reveals an alternative, Pf-indu ...[more]

PMID: 15257299

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Project description:Protein structure can be flexible to adopt multiple conformations to house small molecules. In this paper, we have attempted to experimentally figure out how the structure of a transport protein steer the drug-drug competition (DDC) by maintaining the equilibrium distribution of the bound and unbound fractions of drugs. This understanding is an important facet in biophysical and medicinal chemistry to ascertain the effectiveness of drugs. It is important to note that majority of studies involving small-molecule-transport protein interaction aimed at characterizing the binding process, and because these proteins can interact with thousands of molecules, there are hundreds of reports on such interactions. This ultimately led to an impression among the readers that any studies involving serum albumin may not lead to any new findings except for traditional binding explorations. However, in the present paper, we would like to draw the attention of the readers that our findings are very surprising, new, and important, involving the phenomenon of ligand-protein interaction. Here, we have studied two structurally similar drugs methotrexate (MTX) and folic acid (FA), which attempt to bind the primary binding site (subdomain IIA), one at a time, of human serum albumin. Details of binding analyses reveal that when both of the drugs are present, the single-site binding mode of FA prefers to occupy the primary binding site and hence pushes the primary-site-bound MTX to another location (subdomain IIIA), which is the second binding site of MTX. The structural analysis indicates that DDC has occurred in a cooperative fashion so that the loss of the protein secondary structure is minimum when both drugs are bound to the protein, which means that in the case of duo-drug binding, the conventional interaction between the drug and the protein is altered to undergo restoration of the protein structure. This can indeed regulate the DDC by modifying the bound and unbound fractions of MTX in plasma. The present study emphasizes that in vitro structural characterizations of the transport protein provide important information to improve the molecular-level understanding of DDC for further therapeutic applications with combination drug.

Project description:Biomolecular recognition between proteins follows complex mechanisms, the understanding of which can substantially advance drug discovery efforts. Here, we track each step of the binding process in atomistic detail with molecular dynamics simulations using trypsin and its inhibitor bovine pancreatic trypsin inhibitor (BPTI) as a model system. We use umbrella sampling to cover a range of unbinding pathways. Starting from these simulations, we subsequently seed classical simulations at different stages of the process and combine them to a Markov state model. We clearly identify three kinetically separated states (an unbound state, an encounter state, and the final complex) and describe the mechanisms that dominate the binding process. From our model, we propose the following sequence of events. The initial formation of the encounter complex is driven by long-range interactions because opposite charges in trypsin and BPTI draw them together. The encounter complex features the prealigned binding partners with binding sites still partially surrounded by solvation shells. Further approaching leads to desolvation and increases the importance of van der Waals interactions. The native binding pose is adopted by maximizing short-range interactions. Thereby side-chain rearrangements ensure optimal shape complementarity. In particular, BPTI's P1 residue adapts to the S1 pocket and prime site residues reorient to optimize interactions. After the paradigm of conformation selection, binding-competent conformations of BPTI and trypsin are already present in the apo ensembles and their probabilities increase during this proposed two-step association process. This detailed characterization of the molecular forces driving the binding process includes numerous aspects that have been discussed as central to the binding of trypsin and BPTI and protein complex formation in general. In this study, we combine all these aspects into one comprehensive model of protein recognition. We thereby contribute to enhance our general understanding of this fundamental mechanism, which is particularly critical as the development of biopharmaceuticals continuously gains significance.

Dataset Information

Structural mechanism of the simultaneous binding of two drugs to a multidrug-binding protein.

Publications

Structural mechanism of the simultaneous binding of two drugs to a multidrug-binding protein.

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