A fiducial-assisted strategy compatible with resolving small MFS transporter structures in multiple conformations...

Area of Science:

• Structural Biology
• Biophysics
• Molecular Biology

Background:

• Cryo-electron microscopy (cryo-EM) has advanced structural biology, but challenges remain for small membrane proteins (<40 kDa).
• Determining multiple protein conformations from a single sample is difficult, especially for transporters and G-protein coupled receptors.
• Lack of distinguishable features hinders image alignment and structure determination for these proteins.

Purpose of the Study:

• To develop a strategy for determining high-resolution cryo-EM structures of membrane transporters near the size threshold.
• To enable the characterization of multiple conformational states within a single sample.
• To validate a novel fusion construct approach for cryo-EM studies of Major Facilitator Superfamily (MFS) transporters.

Main Methods:

• Fusion of the target transporter's C-terminus with the BRIL domain using a poly-alanine linker.
• Utilizing AlphaFold2 predictions to guide construct design and minimize steric clash.
• Employing a BRIL-specific Fab fragment for enhanced particle orientation and image alignment.
• Applying cryo-EM to elucidate structures of the Staphylococcus aureus NorA transporter and three other MFS transporters.

Main Results:

• Successfully determined four cryo-EM structures of the 42 kDa NorA transporter.
• Resolved three distinct conformations (inward-open, inward-occluded, occluded) of NorA from a single sample.
• Validated the fusion strategy for three additional MFS transporters (GlpT, Bmr, Blt), confirming the utility of a rigid linker.
• Demonstrated that AlphaFold2 predictions facilitated efficient construct design.

Conclusions:

• The BRIL fusion strategy is effective for cryo-EM structure determination of small membrane transporters, including MFS family members.
• This method facilitates the capture and analysis of diverse protein conformations from a single experiment.
• The approach significantly reduces guesswork and screening, making cryo-EM more accessible for studying membrane protein dynamics.