Probing structural and dynamic features in the mitochondrial oxoglutarate carrier by site directed spin-labeling

The mitochondrial oxoglutarate carrier (OGC) catalyzes the transport of 2-oxoglutarate across the mitochondrial inner membrane in exchange for malate, or other dicarboxylates, and belongs to a large family of related transport proteins called the mitochondrial carrier family (MCF). The primary structures of the family members are made up of three tandemly-repeated homologous domains of about 100 amino acids in length, each containing a characteristic sequence motif and two hydrophobic stretches1. No 3D structure is available for OGC yet, but several spectroscopic and functional studies have unveiled some of its features. An important contribution to the description of the structural characteristics of MCF has been provided by EM studies on two dimensional crystals of yeast ADP/ATP carrier (AAC)2 and by elucidation of the crystal structure of bovine AAC3. The OGC seems to be folded into 6 transmembrane segments too in the inner mitochondrial membrane. Site directed spin-labeling (SDSL) has been used to probe the structural and dynamic features of residues comprising the sixth transmembrane segment of the mitochondrial oxoglutarate carrier. Starting from a functional carrier, where cysteines have been replaced by serines, 18 consecutive residues (from G281 to I298) have been mutated to cysteine and subsequently labeled with a thiol-selective nitroxide probe. The labeled proteins, reconstituted into liposomes, have been assayed for their transport activity and analyzed with continuous-wave electron paramagnetic resonance. Linewidth analysis, that is correlated to local probe mobility, indicates a well defined periodicity of the whole segment from G281 to I298, indicating that it has an ahelical structure. Saturation behaviour, in presence of paramagnetic perturbants of different hydrophobicities, allow the definition of the polarity of the individual residues and to assign their orientation with respect to the lipid bilayer or to the water accessible translocation channel. Comparison of the EPR data, homology model and activity data indicate that the segment is made by an a-helix, accommodated in an amphipathic environment, partially distorted in the middle at the level of L289, probably because of the presence of a proline residue (P291). The C-terminal region of the segment is less restrained and more flexible than the N-terminus. This work puts together another piece of the puzzle in the quest for elucidating the structure-function relationship of the mitochondrial oxoglutarate carrier.