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Research Themes Membrane proteins

Membrane Proteome: Building a Carrier

SBKB [doi:10.1038/sbkb.2012.107]
Featured Article - November 2012
Short description: NMR methods and molecular fragment searching are used to generate a structural model of a mitochondrial carrier protein.

Ensemble of 15 UCP2 structures obtained using RDC and PRE restraints. 1

The pumping of protons from the mitochondrial matrix to the intermembrane space, a task achieved by the mitochondrial respiratory complex, builds up an electrochemical potential. Some of this potential is dissipated by the action of mitochondrial uncoupling proteins (UCPs), integral membrane proteins that leak protons back to the matrix. UCPs require fatty acid cofactors to translocate protons and are inhibited by GDP, but the mechanism by which translocation occurs is unknown.

To address this question, Chou and colleagues (PSI MPSbyNMR) used solution NMR methodologies to determine the structure of UCP2. After attempting to solve its structure using traditional NOE-based solution NMR methods, the authors used a method called molecular fragment replacement. The PDB was searched for fragments of structure that agree with experimental restraints derived from residual dipolar couplings (RDCs) collected from detergent-solubilized UCP2 in DNA nanotubes. The authors were able to identify 15 structured segments, derived from water-soluble and membrane-associated proteins, which agreed with the RDC data. A tertiary structure was built using the orientational restraints from the RDCs and paramagnetic relaxation enhancement (PRE)-derived distance restraints.

The final model for UCP2 (PDB 2LCK) is quite similar to the structure of the bovine ADP/ATP carrier ANT1 (PDB 1OKC), another member of the mitochondrial anion carrier protein family. Like ANT1, UCP2 has a channel-like structure formed by three pseudo-repeats. Each repeat consists of a transmembrane helix, a loop and an amphipathic helix, followed by a second transmembrane helix. PRE data using labeled GDP shows that it binds deep within the UCP2 channel. A comparison with the ANT1 structure shows a different orientation of the amphipathic helix in the third repeat of UCP2 that causes a break in the three-fold pseudo-symmetry. This and related structural differences make UCP2 more open on the matrix side of the channel. It remains to be seen if these structural differences play a functional role.

While additional structures of carriers in different functional states will be needed to fully understand the mechanism of transport and substrate selectivity, the work described here supports the idea that this carrier family has a largely conserved fold and that minor variations within the fold are responsible for substrate specificity. The authors suggest that RDCs would be useful in monitoring changes in structural orientation linked to different functional states.

Michelle Montoya

References

  1. M.J. Berardi et al. Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching.
    Nature. 476, 109-113 (2011). doi:10.1038/nature10257

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