Experimentally optimized threading structures of the proton-coupled folate transporter

Area of Science:

• Structural biology
• Membrane transport
• Biochemistry

Background:

• The proton-coupled folate transporter (PCFT, SLC46A1) mediates folic acid uptake via proton symport.
• Understanding PCFT's structure is crucial for elucidating its transport mechanism and substrate binding.
• PCFT belongs to the Major Facilitator Superfamily (MFS), sharing conserved structural motifs.

Purpose of the Study:

• To develop and validate 3D structural models of PCFT based on homologous MFS transporters.
• To investigate the structural basis of PCFT's proton-coupled transport mechanism.
• To correlate structural models with existing functional and mutagenesis data.

Main Methods:

• Development of 3D structural models of PCFT by threading onto X-ray structures of MFS members (e.g., GlpT).
• Consistency checks using accessibility studies at pH 7.4 and in the absence of substrate.
• Integration of mutagenesis and functional data to refine the PCFT model.

Main Results:

• A validated 3D model of PCFT was generated, preserving characteristic MFS features like 12 transmembrane helices.
• The model is consistent with an inward-open resting state and explains interactions with lipid head-groups.
• Key structural elements, including Motif A and a charge-relay system, were identified and implicated in conformational changes.

Conclusions:

• The developed PCFT structural model provides a framework for understanding its proton-coupled transport mechanism.
• The model successfully integrates diverse experimental data, highlighting the importance of MFS structural features.
• Further studies can utilize this model to investigate substrate binding, proton relay, and conformational transitions.