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Research Themes Membrane proteins

A fragmented approach to membrane protein structures

SBKB [doi:10.1038/sbkb.2011.38]
Technical Highlight - September 2011
Short description: An innovative method combines NMR experiments and a database search to determine the structure of a challenging membrane protein.

Side view of UCP2 as determined by Chou and colleagues. Reprinted from Nature 1 .

Membrane protein structure determination by NMR has advanced considerably owing to developments in sample preparation and experiments for large proteins solubilized in model membranes. Usually, the primary goal is nuclear Overhauser enhancement (NOE) data, which yield distance restraints of ∼5 Å or less and constitute the classical method of NMR structure determination. Newer techniques provide essential complementary data. Specifically, residual dipolar coupling (RDC) data report on local bond orientations, whereas paramagnetic relaxation enhancement (PRE) data provide long-range distance restraints up to ∼25 Å.

Chou and colleagues (PSI MPSbyNMR) have used such data to characterize UCP2, a member of the mitochondrial carrier protein family. Severe signal overlap precluded NOE-based structure determination, but by weakly orienting UCP2 in DNA nanotubes the authors obtained 2.2 RDC restraints per residue. Although three or more RDCs per residue are usually required to solve structures, the authors successfully adapted the molecular fragment replacement (MFR) method previously used for crystallography. By comparing a database of 320,000 fragments from the Protein Data Bank with the RDC data, they identified 15 segments covering most of the UCP2 sequence. They next measured 452 PRE restraints using four nitroxide-labeled samples. A two-stage procedure first used the MFR fragments to calculate local coordinates and then introduced the PRE restraints to determine the spatial arrangement of the fragments in the presence of GDP, a known inhibitor. The calculations resulted in a structure ensemble with a backbone precision of 1.3 Å.

Although the authors caution that no data were available for the side chains, they could conclude that UCP2 is a structural homolog of the ADP/ATP carrier ANT1, despite having only ∼20% sequence identity. Furthermore, they found that GDP binds in the UCP2 channel similarly to how ADP may bind ANT1. Comparison of the transmembrane helix topology shows that the UCP2 channel is considerably more open than in ANT1. Members of the carrier family may have evolved a conserved global structure with localized fold variations conferring substrate selectivity. As more than 40 types of carriers are known, the method promises to be applicable to structural analysis of different functional states of membrane proteins.

Michael A. Durney

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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