Structural models of mitochondrial uncoupling proteins obtained in DPC micelles are not functionally relevant

Area of Science:

• Mitochondrial physiology
• Membrane protein biochemistry
• Brown adipose tissue function

Background:

• Uncoupling protein 1 (UCP1) in brown adipocytes regulates energy expenditure via proton conductance and fatty acid oxidation.
• Previous atomic models of UCP1/UCP2 were based on NMR structures in dodecylphosphocholine (DPC), a detergent known to inhibit UCP1.
• NMR studies suggested Lysine 56 (K56) and Lysine 269 (K269) are vital for long-chain fatty acid (LCFA) binding and UCP1 activation.

Purpose of the Study:

• To re-evaluate the role of K56 and K269 in UCP1 function using a more physiological system.
• To investigate the relevance of previous findings obtained in detergent (DPC) to UCP1's biological activity.
• To determine if K56 and K269 are essential for LCFA-induced UCP1 activation in vivo.

Main Methods:

• Expression of UCP1 mutants (K56S, K269S, K56S/K269S) in Saccharomyces cerevisiae mitochondria.
• Assay of mitochondrial respiration in permeabilized spheroplasts to measure UCP1 activation and inhibition.
• Utilizing a physiological context to assess UCP1 structure-function relationships.

Main Results:

• UCP1 mutants K56S, K269S, and K56S/K269S showed no defects in activation in the yeast mitochondria.
• The physiological assays confirmed UCP1 activation and inhibition capabilities in the engineered yeast model.
• These results contradict the hypothesis derived from NMR titration experiments in DPC.

Conclusions:

• The residues K56 and K269 are not crucial for UCP1 activation by LCFAs in a physiological context.
• NMR titration studies performed in DPC detergent do not accurately reflect UCP1 function in a biological membrane.
• This study highlights the limitations of detergent-based structural analyses for membrane proteins like UCP1 and emphasizes the need for in vivo validation.