Single-molecule dynamics reveal ATP binding alone powers substrate translocation by an ABC transporter

Area of Science:

• Biochemistry
• Molecular Biology
• Immunology

Background:

• ATP-binding cassette (ABC) transporters are crucial for cellular functions.
• The mechanism linking ATP binding to substrate translocation in ABC transporters remains unclear.
• TAP transporters are vital for adaptive immunity through antigen processing.

Purpose of the Study:

• To elucidate the mechanism of substrate translocation in ABC transporters at the single-molecule level.
• To investigate the role of ATP binding and hydrolysis in ABC transporter function.
• To understand the conformational changes driving peptide transport.

Main Methods:

• Single-molecule Förster Resonance Energy Transfer (smFRET) to visualize individual translocation events.
• Utilizing a slow-turnover variant of the bacterial homolog TmrAB.
• Cryo-electron microscopy (Cryo-EM) to determine transporter structures.

Main Results:

• Substrate transport is driven by a conformational switch from inward- to outward-facing states.
• ATP binding alone, without Mg²⁺, can drive a single round of peptide translocation.
• Cryo-EM structures confirm ATP binding induces the outward-facing conformation.
• Mg²⁺-dependent ATP hydrolysis is necessary for resetting the transporter after translocation.

Conclusions:

• A direct mechanistic link between ATP binding and substrate translocation is established at single-molecule resolution.
• The findings provide insights into the catalytic cycle of ABC transporters.
• This study clarifies the distinct roles of ATP binding and hydrolysis in transporter function.