Capturing transient states of heterodimeric ABC transporter TM287/288 by time-resolved small-angle X-ray scattering

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biophysics

Background:

• The heterodimeric ABC transporter TM287/288 undergoes significant conformational changes during its functional cycle.
• Static structural biology methods have captured several states but miss transient intermediates.
• A complete understanding of the TM287/288 reaction cycle requires characterization of these missing transient states, particularly occluded states.

Purpose of the Study:

• To investigate the kinetics and structural dynamics of the full-length TM287/288 transporter during its ATP-driven cycle.
• To identify and characterize transient states within the transporter's reaction mechanism.
• To provide a more complete description of the conformational changes involved in ABC transporter function.

Main Methods:

• Utilized time-resolved small-angle X-ray scattering (TR-SAXS) initiated by stopped-flow mixing.
• Employed active site mutants of TM287/288 to probe specific steps in the reaction cycle.
• Leveraged state-specific sybodies/nanobodies to stabilize and identify different conformational states.

Main Results:

• Dissected the temporal events governing the ATP-driven conformational cycle of TM287/288.
• Successfully captured and characterized a previously unobserved transient occluded state.
• Provided kinetic and structural insights into the complete reaction mechanism of this ABC transporter.

Conclusions:

• The study successfully identified a transient occluded state in the TM287/288 ABC transporter's reaction cycle.
• Time-resolved SAXS combined with mutagenesis and nanobodies is effective for studying dynamic biological processes.
• This work significantly advances the understanding of ABC transporter mechanisms and conformational dynamics.