Identification of the Substrate Binding Sites within the Yeast Mitochondrial Citrate Transport Protein

Abstract

The objective of the present investigation was to identify the substrate binding site(s) within the yeast mitochondrial citrate transport protein (CTP). Our strategy involved kinetically characterizing 30 single-Cys CTP mutants that we had previously constructed based on their hypothesized importance in the structure-based mechanism of this carrier. As part of these studies, a modified transport assay was developed that permitted, for the first time, the accurate determination of K m values that were elevated >100-fold compared with the Cys-less control value. We identified 10 single-Cys CTP mutants that displayed sharply elevated K m values (i.e. 5 to >300-fold). Each of these mutants displayed V max values that were reduced by >98% and resultant catalytic efficiencies that were reduced by >99.9%. Importantly, superposition of this functional data onto the three-dimensional homology-modeled CTP structure, which we previously had developed, revealed that nine of these ten residues form two topographically distinct clusters. Additional modeling showed that: (i) each cluster is capable of forming numerous hydrogen bonds with citrate and (ii) the two clusters are sufficiently distant from one another such that citrate is unlikely to interact with all of these residues at the same time. We deduced from these findings that the CTP contains at least two citrate binding sites per monomer, which are located at increasing depths within the translocation pathway. The identification of these sites, combined with an initial assessment of the citrate-amino acid side-chain interactions that may occur at these sites, substantially extends our understanding of CTP functioning at the molecular level.The mitochondrial citrate transport protein (CTP) 3 is located within the inner mitochondrial membrane and catalyzes an obligatory exchange of the dibasic form of tricarboxylic acids (e.g. citrate and isocitrate) for other tricarboxylic acids or in higher eukaryotes for dicarboxylic acids (e.g. malate and succinate) or phosphoenolpyruvate (1). Once in the cytoplasm, the transported citrate serves as the prime carbon source fueling fatty acid, triacylglycerol, and cholesterol biosyntheses (2-5). In addition, the concerted action of citrate lyase and malate dehydrogenase enables the generation of NAD ϩ , a cofactor that is essential for the glycolytic pathway. Based on these roles, the CTP is considered essential for eukaryotic cell metabolism.Because of the prominent role of the CTP in cellular bioenergetics, our laboratory has conducted extensive investigations with the aim of elucidating its structure-based mechanism. Thus we have cloned (6), overexpressed (7,8), and purified (9, 10) the functional form of this transporter. Recently, employing a Cys-less yeast mitochondrial CTP construct that displays native functional properties (11) as the template, we have: (i) demonstrated that the transporter exists as a homodimer in detergent micelles (12); (ii) utilized cysteine-scanning mutagenesis combined with probing the accessibility o...

Severe Neonatal Presentation of Mitochondrial Citrate Carrier (SLC25A1) Deficiency

“…In the orthologous S. cerevisiae citrate transport protein CTP, the equivalent position (Arg189) is proposed to form direct physical contacts with citrate at one of the protein's two putative citrate-binding sites (Ma et al 2007). Substitution of Arg189 with a cysteine drastically compromises both the kinetics and the selectivity of CTP, with a >300-fold increase in K m ,~1,000-fold decrease in V max , and a greatly increased affinity for dicarboxylic acids (e.g., malate) rather than citrate (Ma et al 2007;Aluvila et al 2010). An in silico structural model based on the homologous mitochondrial carrier ANT1 (PDB: 10KC) (Pebay-Peyroula et al 2003) situates the side chain of Arg198 within CIC's positively charged site 1 pocket (Supplemental Fig.…”

The mitochondrial citrate carrier: Metabolic role and regulation of its activity and expression

“…The superimposition with functional data of the three-dimensional homology model, developed on the X-ray structure of the mitochondrial ADP/ATP carrier (44), allowed to clearly identify substantial portions of the substrate translocation pathway through the transmembrane domains III and IV of CiC, also confirming their a-helical structure (45)(46)(47). Furthermore, transport kinetic studies conducted with a panel of singleCys CiC yeast mutants, in combination with molecular modeling studies, have permitted the clear-cut identification of two substrate binding sites per monomer within CiC that reside at increasing depths in the bilayer (48). The composition of these binding sites highlights the importance of lysine and arginine residues in the binding of the substrate (i.e., citrate).…”

Inhibitors of the Mitochondrial Citrate Transport Protein: Validation of the Role of Substrate Binding Residues and Discovery of the First Purely Competitive Inhibitor

“…Using a combination of docking calculations and analysis of the transport kinetics of single point mutations, we have identified two substrate binding sites in this transporter in its cytosolic-facing conformation (Ma et al, 2007) as depicted in Fig. 1.…”