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. 2008 Nov 18;105(46):17766-71.
doi: 10.1073/pnas.0809580105. Epub 2008 Nov 10.

The mechanism of transport by mitochondrial carriers based on analysis of symmetry

Alan J Robinson  1 , Catherine Overy, Edmund R S Kunji
Affiliations

The mechanism of transport by mitochondrial carriers based on analysis of symmetry

Alan J Robinson et al. Proc Natl Acad Sci U S A. .

Abstract

The structures of mitochondrial transporters and uncoupling proteins are 3-fold pseudosymmetrical, but their substrates and coupling ions are not. Thus, deviations from symmetry are to be expected in the substrate and ion-binding sites in the central aqueous cavity. By analyzing the 3-fold pseudosymmetrical repeats from which their sequences are made, conserved asymmetric residues were found to cluster in a region of the central cavity identified previously as the common substrate-binding site. Conserved symmetrical residues required for the transport mechanism were found at the water-membrane interfaces, and they include the three PX[DE]XX[RK] motifs, which form a salt bridge network on the matrix side of the cavity when the substrate-binding site is open to the mitochondrial intermembrane space. Symmetrical residues in three [FY][DE]XX[RK] motifs are on the cytoplasmic side of the cavity and could form a salt bridge network when the substrate-binding site is accessible from the mitochondrial matrix. It is proposed that the opening and closing of the carrier may be coupled to the disruption and formation of the 2 salt bridge networks via a 3-fold rotary twist induced by substrate binding. The interaction energies of the networks allow members of the transporter family to be classified as strict exchangers or uniporters.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Scoring of symmetry in the yeast mitochondrial phosphate transporter ScPic2p. (A) The alignment of the even-numbered α-helices in ScPic2p. Highlighted in color are 2 examples of symmetry-related triplets of residues, as used in B and C. (B and C) The replacement scores (black) for each comparison in the triplet were obtained from the residue replace ability matrix (55). The symmetry score of a residue (green) is the average of the 2 replacement scores. (B) Triplet E-E-D with symmetric residues. (C) Triplet K-V-K with an asymmetric valine residue. (D) The cumulative frequency distribution of the symmetry scores for all possible triplets in the sequences of the subfamily of fungal Pic2p transporters. The symmetry scores are defined by a red-white-blue color scale, in which the median of the distribution is white and the minimal and maximal symmetry scores red and blue, respectively (Fig. 2 A and B). The symmetry scores of D288 (B) and V191 (C) (conservation scores in brackets) are shown.
Fig. 2.
Fig. 2.
Asymmetry and symmetry in fungal phosphate transporters and mammalian uncoupling proteins. Average symmetry and conservation scores in the subfamily of (A and B) the phosphate transporters and (D and E) the uncoupling proteins projected onto a model of the bovine ADP/ATP transporter (8). The positive conservation score and average symmetry score are represented by the size and color of the Cβ atom, respectively (Fig. 1D). (A and D) Asymmetric residues with negative average symmetry scores. Residues in the cavity are labeled. (B and E) Symmetric residues with positive average symmetry scores. Highly symmetric residues are labeled. Dashed circles and rectangles indicate the location of the substrate binding site and networks, respectively. Green spheres represent residues that are absent from the repeat. Also shown are V191 and D288 (Fig. 1D). For all analyzed sequences the average of the standard deviations for all of the average symmetry scores of the cavity residues was 0.10. (C and F) The residues of the cytoplasmic (Upper) and matrix network (Lower) in ScPic2p and HsUCP1, respectively. The positively and negatively charged residues of the salt bridges are shown in blue and red, respectively. Deviating polar, aromatic and apolar residues are shown in green, orange and pink, respectively. The interaction energies of the network are quantified as the number of salt bridges, taking hydrogen bonds and cation-π interactions as half the interaction energy of a salt bridge, and van der Waals interactions as negligible.
Fig. 3.
Fig. 3.
The substrate-binding site and salt bridge networks of the mitochondrial ADP/ATP carrier. An ADP molecule and a comparative model of the yeast ADP/ATP carrier ScAac2p (12) are shown in ball/stick and diagram representation, respectively. The residues involved in substrate binding are shown in red and the contact points of the common substrate-binding site are indicated by black circles and Roman numerals (12). The residues of the matrix and the cytoplasmic network are shown in green and blue, respectively.
Fig. 4.
Fig. 4.
Dependency of the transport mode on the interaction energies of the salt bridge networks. Transport cycles showing (A) strict equimolar exchange and (B) uniport. The contact points of the common substrate binding site are shown as black spheres with Roman numerals. Negatively charged, positively charged and polar residues of the networks are shown as red, blue and green sticks, respectively. The exported and imported substrates are green and cyan, respectively. All transport steps are fully reversible, but the direction of transport (red arrows) is determined by the concentration gradients of the substrates and the membrane potential in the case of electrogenic transport. During substrate import, the matrix network on the odd-numbered α-helices is released, whereas the cytoplasmic network on the even-numbered α-helices is formed, and the reverse during substrate export. The structural changes might occur with 3-fold pseudosymmetry via a rotary twist (orange arrows).
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