Mapping the nucleotide binding site of uncoupling protein 1 using atomic force microscopy

Area of Science:

• Mitochondrial physiology
• Membrane protein function
• Biophysics

Background:

• Proton transport via uncoupling proteins (UCPs) is vital for ATP synthesis, heat production, and reactive oxygen species regulation.
• Dysregulation of UCPs is implicated in obesity, inflammation, neurodegeneration, and ischemia.
• Purine nucleotides, particularly ATP, inhibit UCP1 and UCP2, but the precise mechanism and binding site remain unclear, especially given high intramitochondrial ATP concentrations.

Purpose of the Study:

• To investigate the structural basis of ATP interaction with UCP1 at a single-molecule level.
• To determine the accessibility and precise location of the ATP binding site on UCP1 within the inner mitochondrial membrane.

Main Methods:

• Utilized the topographic and recognition (TREC) mode of atomic force microscopy (AFM).
• Reconstituted purified UCP1 into lipid bilayers for AFM analysis.
• Employed anti-UCP1 antibody and ATP for recognition pattern analysis.
• Used AFM cantilever tips with varying cross-linker lengths to achieve angstrom-level precision.

Main Results:

• Visualized UCP1 within lipid bilayers and analyzed ATP-protein interactions at the single-molecule level.
• Demonstrated that the ATP binding site on UCP1 is accessible from both sides of the mitochondrial membrane.
• Precisely localized the nucleotide binding site within the membrane with 1 Å resolution.

Conclusions:

• The study provides crucial structural insights into how ATP binds to UCP1.
• Findings suggest a mechanism for UCP1 function despite high intramitochondrial ATP levels.
• This research lays the groundwork for developing novel pharmacological strategies targeting UCPs for various diseases.