ATP-driven conformational dynamics reveal hidden intermediates in a heterodimeric ABC transporter

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• ATP-binding cassette (ABC) transporters are crucial molecular machines.
• Their conformational dynamics are typically studied using ensemble-averaged methods.
• Single-molecule techniques are needed to resolve dynamic heterogeneity and transient intermediates.

Purpose of the Study:

• To resolve ATP-driven conformational dynamics of the heterodimeric type IV ABC transporter TmrAB using single-molecule Förster resonance energy transfer (smFRET).
• To characterize the functional homolog of the human antigen transporter TAP at the single-molecule level.
• To establish a quantitative single-molecule framework for dissecting ATP-coupled conformational dynamics in heterodimeric ABC transporters.

Main Methods:

• Single-molecule Förster resonance energy transfer (smFRET) was employed to study TmrAB.
• Fluorophore placement at nucleotide-binding domains and periplasmic gate was validated using accessible-volume simulations, fluorescence lifetimes, and ensemble FRET.
• Kinetic analysis and complementary stabilization strategies were utilized.

Main Results:

• Single-molecule analysis successfully distinguished ATP-free and ATP-bound states of TmrAB.
• ATP-dependent population shifts were quantified under varying nucleotide concentrations.
• An unexpectedly long ATP-bound dwell time of approximately 300 ms was observed.
• A previously hidden outward-facing open state, kinetically masked under turnover, was directly resolved.

Conclusions:

• This study provides the first single-molecule characterization of the TmrAB transporter.
• The findings offer a quantitative single-molecule framework for understanding conformational dynamics in heterodimeric ABC transporters.
• The resolution of a hidden outward-facing state advances the understanding of transporter mechanisms.