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3D model of the Escherichia coli multidrug transporter MdfA reveals an essential membrane-embedded positive charge
Nadejda Sigal1, Eyal Vardy, Shahar Molshanski-Mor
1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Abstract:
MdfA is an Escherichia coli multidrug transporter of the major facilitator superfamily (MFS) of secondary transporters. Although several aspects of multidrug recognition by MdfA have been characterized, better understanding the detailed mechanism of its function requires structural information. Previous studies have modeled the 3D structures of MFS proteins, based on the X-ray structure of LacY and GlpT. However, because of poor sequence homology, between LacY, GlpT, and MdfA additional constraints were required for a reliable homology modeling. Using an algorithm that predicts the angular orientation of each transmembrane helix (TM) (kPROT), we obtained a remarkably similar pattern for the 12 TMs of MdfA and those of GlpT and LacY, suggesting that they all have similar helix packing. Consequently, a 3D model was constructed for MdfA by structural alignment with LacY and GlpT, using the kPROT results as an additional constraint. Further refinement and a preliminary evaluation of the model were achieved by correlated mutation analysis and the available experimental data. Surprisingly, in addition to the previously characterized membrane-embedded glutamate at position 26, the model suggests that Asp34 and Arg112 are located within the membrane, on the same face of the cavity as Glu26. Importantly, Arg112 is evolutionarily conserved in secondary drug transporters, and here we show that a positive charge at this position is absolutely essential for multidrug transport by MdfA.
Insights
Structural insights into the Escherichia coli MdfA multidrug transporter were gained using homology modeling. A conserved positively charged residue, Arginine 112, was found essential for MdfA
Area of Science:
- Structural biology
- Molecular microbiology
- Biochemistry
Background:
- MdfA is an Escherichia coli multidrug transporter belonging to the major facilitator superfamily (MFS).
- Understanding MdfA's transport mechanism requires detailed structural information, which has been challenging due to low sequence homology with existing MFS structural models.
- Previous homology modeling efforts for MFS proteins relied on structures like LacY and GlpT, necessitating additional constraints for MdfA.
Purpose of the Study:
- To generate a reliable 3D structural model of the MdfA multidrug transporter.
- To investigate the structural basis of MdfA's function and identify key residues involved in multidrug transport.
Main Methods:
- Utilized the kPROT algorithm to predict transmembrane helix angular orientation, providing constraints for homology modeling.
- Performed structural alignment of MdfA with LacY and GlpT, incorporating kPROT predictions.
- Refined the MdfA model using correlated mutation analysis and available experimental data.
Main Results:
- The kPROT algorithm revealed similar transmembrane helix packing patterns between MdfA, GlpT, and LacY.
- A 3D model of MdfA was constructed, suggesting the presence of membrane-embedded residues Asp34 and Arg112 within the transport cavity, alongside Glu26.
- Demonstrated that a positive charge at position Arg112 is critical for MdfA's multidrug transport activity.
Conclusions:
- The developed 3D model provides valuable structural insights into MdfA function.
- The evolutionary conservation and functional necessity of Arg112 highlight its crucial role in MdfA-mediated multidrug transport.
- This study enhances the understanding of MFS transporter mechanisms and drug resistance in bacteria.
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