The mitochondrial carnitine/acylcarnitine carrier: structure/function relationship by site-directed mutagenesis and homology modeling.

To study the mitochondrial carnitine acylcarnitine carrier (CAC) structural-functional relationships by site directed mutagenesis, we over expressed it in E. coli (1); the over expressed protein showed the same functional properties of the native carrier. The role of Cys residues of the carnitine carrier have been investigated substituting Cys residues with Ser. Inhibition analysis of single and multiple Cys mutants revealed that C136, the major target for SH reagents, is located at or near the substrate binding site. The effect of SH oxidizing, cross linking and coordinating reagents was evaluated on the carnitine/ carnitine exchange catalyzed by reconstituted multiple Cys mutants of the CAC. All the reagents tested efficiently inhibited the wild type CAC. The inhibitory effect of diamide, Cu-phenanthroline or phenylarsine oxide was largely reduced or abolished in the mutants in which two of the three residues C58, C136 and C155 had been substituted with Ser. Sb, which coordinates three Cys, inhibited only the Cys replacement mutants containing C58, C136 and C155. The results described demonstrate that C58, C136 and C155 are close each other in the tertiary structure of CAC. These results, were interpreted in the light of the homology model of CAC constructed by using the X-ray structure of the ADP ATP carrier (2) as template. In this model C58, C136 and C155 are clustered at approximately identical distances from the membrane aqueous interface near the mitochondrial matrix side. However, the calculated distances between the side chains of these Cys (9.4 A and 14.8 A between C136C155 and C58C136, respectively) are greater than those required for the observed chemical rnodifications. This means that C58, C136 and C155 become closer during one or more steps of the catalytic cycle. The presence of the substrate carnitine, inside and outside the proteoliposomes, induces conformational changes of the protein and thereby facilitates the cross linking of the Cys pairs. 1. Indiveri, C., lacobazzi, V., Giangregorio, N. and Palmieri, F. (1998) Biochem. Biophys. Res. Commun. 249, 589-594 2. Pebay-Peyroula, E., Dahout-Gonzalez, C., Kahn, R., Trezeguet, V., Lauquin, G.J., and Brandolin, G. (2003) Nature 426,39-44