Mitochondrial uncoupling protein 2 structure determined by NMR molecular fragment searching

Abstract

Mitochondrial uncoupling protein 2 (UCP2) is an integral membrane protein in the mitochondrial anion carrier protein family, the members of which facilitate the transport of small molecules across the mitochondrial inner membrane. When the mitochondrial respiratory complex pumps protons from the mitochondrial matrix to the intermembrane space, it builds up an electrochemical potential. A fraction of this electrochemical potential is dissipated as heat, in a process involving leakage of protons back to the matrix. This leakage, or 'uncoupling' of the proton electrochemical potential, is mediated primarily by uncoupling proteins. However, the mechanism of UCP-mediated proton translocation across the lipid bilayer is unknown. Here we describe a solution-NMR method for structural characterization of UCP2. The method, which overcomes some of the challenges associated with membrane-protein structure determination, combines orientation restraints derived from NMR residual dipolar couplings (RDCs) and semiquantitative distance restraints from paramagnetic relaxation enhancement (PRE) measurements. The local and secondary structures of the protein were determined by piecing together molecular fragments from the Protein Data Bank that best fit experimental RDCs from samples weakly aligned in a DNA nanotube liquid crystal. The RDCs also determine the relative orientation of the secondary structural segments, and the PRE restraints provide their spatial arrangement in the tertiary fold. UCP2 closely resembles the bovine ADP/ATP carrier (the only carrier protein of known structure), but the relative orientations of the helical segments are different, resulting in a wider opening on the matrix side of the inner membrane. Moreover, the nitroxide-labelled GDP binds inside the channel and seems to be closer to transmembrane helices 1-4. We believe that this biophysical approach can be applied to other membrane proteins and, in particular, to other mitochondrial carriers, not only for structure determination but also to characterize various conformational states of these proteins linked to substrate transport.

Figure 1. NMR spectra, GDP binding, and outline of RDC-based molecular fragment assignment

a, The ¹H^N-¹⁵N TROSY HSQC spectrum of ¹⁵N-,¹³C-,²H-labelled UCP2 reconstituted in DPC micelles (with 5 mM GDP) recorded at pH 6.5, 33 °C, and ¹H frequency of 600 MHz. b, The (¹⁵N,¹³C’) projection of the 3D TROSY HNCO spectrum of the sample in a, recorded under the same conditions. Comparison of a and b, illustrates that most resonances can be resolved in the 3D HNCO spectrum. c, Specific binding of GDP to UCP2 under conditions of the NMR sample. The FRET Response (= %[FU_{MANT-GDP / GDP} - FU⁰_GDP]; see METHODS) follows displacement of fluorescent MANT-GDP by GDP as the concentration of the latter increases. d, Histogram of 470 unambiguously assigned ¹D_NH, ¹D_C’Cα or ¹D_NC’ (all normalized to ¹D_NH). The magnitude (D_a) and rhombicity (R_h) of the alignment tensors are 10 Hz and 0.61, respectively.

Figure 2. Conceptual illustration of the operations involved in RDC-based structural segment building

a, Initial fragment assignment. b, gap filling (left) and end extension (right). c, The 15 continuous structured segments of UCP2 determined by RDC-based MFR (shaded and labelled). Details of these operations are described in text and in METHODS.

Figure 3. Solution structure of UCP2 and region of GDP binding

a, UCP2 sequence and membrane topology, with basic and acidic residues shown in blue and red, respectively. The conserved prolines at the proline kinks of TMH 1, 3, and 5 are shown in yellow. The spin-labeled positions are highlighted in green. The red dashed lines represent long-range or inter-helical PRE distances (< 19 Å) between the spin-label and backbone amide protons. b, An ensemble of 15 low-energy structures derived from NMR restraints. The backbone and heavy atom r.m.s. deviations for the structured segments in Fig. 2e are 1.2 Å and 1.8 Å, respectively. c, Chemical structure of the spin-labeled GDP, with the paramagnetic nitroxide moiety circled in red. d, Mapping the effect of spin-labeled GDP onto the ribbon drawing of UCP2. The color gradient is from yellow (resonance intensity ratio of broadened to non-broadened, ε = 1.0) to white (ε = 0.8) to blue (ε = 0.3).

Figure 4. Comparison of UCP2 and ANT1

a, Side views of UCP2 and ANT1 (PDB code: 1OKC). Pseudo-repeats 1, 2, and 3 are in blue, green and pink, respectively. The three repeats are 1: 14–112, 2: 113–210, and 3: 211–309 (see Fig. 3a above for reference). b, Views of UCP2 and ANT1 from the matrix side of the carriers, showing loss of 3-fold pseudo-symmetry in UCP2 as a result of structural differences in repeat 3. The orientations of the amphipathic helices are emphasized by the arrows.