Rusudan Kotaria

Oligomeric State of Wild-Type and Cysteine-Less Yeast Mitochondrial Citrate Transport Proteins

Experiments have been conducted to determine the oligomeric state of the mitochondrial citratetransport protein (CTP) from the yeast Saccharomyces cerevisiae. Both wild-type andcysteine-less (Cys-less) CTPs were overexpressed in E. coli and solubilized with sarkosyl. The purity ofthe solubilized material is approximately 75%. Upon incorporation into phospholipid vesicles, ahigh specific transport activity is obtained with both the wild-type and Cys-less CTPs, therebydemonstrating the structural and functional integrity of the preparations. Two independentapproaches were utilized to determine native molecular weight. First, CTP molecular weightwas determined via nondenaturing size-exclusion chromatography. With this methodology weobtained molecular weight values of 70,961 and 70,118 for the wild-type and Cys-less CTPs,respectively. Second, charge-shift native gel electrophoresis was carried out utilizing a lowconcentration of the negatively charged detergent sarkosyl, which served to both impart acharge shift to the CTP and the protein standards, as well as to promote protein solubility.Via the second method, we obtained molecular weight values of 69,122 and 74,911 forthe wild-type and Cys-less CTPs, respectively. Both methods clearly indicate that followingsolubilization, the wild-type and the Cys-less CTPs exist exclusively as dimers. Furthermore,disulfide bonds are not required for either dimer formation or stabilization. The dimericstate of the CTP has important implications for the structural basis underlying the CTPtranslocation mechanism.

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

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 Km values that were elevated >100-fold compared with the Cys-less control value. We identified 10 single-Cys CTP mutants that displayed sharply elevated Km values (i.e. 5 to >300-fold). Each of these mutants displayed Vmax 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 yeast mitochondrial citrate transport protein: molecular determinants of its substrate specificity.

The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residues comprising the more external site, the CTP becomes less selective for citrate with numerous external anions able to effectively inhibit [14C]citrate/citrate exchange. Thus, the site 1 variants assume the binding characteristics of a nonspecific anion carrier. Comparison of [14C]citrate uptake in the presence of various internal anions versus water revealed that, with the exception of the R189C mutant, the other site 1 variants showed substantial uniport activity relative to exchange. Upon mutation of residues comprising site 2, we observed two types of effects. The K37C mutant displayed a markedly enhanced selectivity for external citrate. In contrast, the other site 2 mutants displayed varying degrees of relaxed selectivity for external citrate. Examination of internal substrates revealed that, in contrast to the control transporter, the R181C variant exclusively functioned as a uniporter. This study provides the first functional information on the role of specific binding site residues in determining mitochondrial transporter substrate selectivity. We interpret our findings in the context of our homology-modeled CTP as it cycles between the outward-facing, occluded, and inward-facing states.

The yeast mitochondrial citrate transport protein: identification of the Lysine residues responsible for inhibition mediated by Pyridoxal 5′-phosphate

The present investigation identifies the molecular basis for the well-documented inhibition of the mitochondrial inner membrane citrate transport protein (CTP) function by the lysine-selective reagent pyridoxal 5′-phosphate. Kinetic analysis indicates that PLP is a linear mixed inhibitor of the Cys-less CTP, with a predominantly competitive component. We have previously concluded that the CTP contains at least two substrate binding sites which are located at increasing depths within the substrate translocation pathway and which contain key lysine residues. In the present investigation, the roles of Lys-83 in substrate binding site one, Lys-37 and Lys-239 in substrate binding site two, and four other off-pathway lysines in conferring PLP-inhibition of transport was determined by functional characterization of seven lysine to cysteine substitution mutants. We observed that replacement of Lys-83 with cysteine resulted in a 78% loss of the PLP-mediated inhibition of CTP function. In contrast, replacement of either Lys-37 or Lys-239 with cysteine caused a modest reduction in the inhibition caused by PLP (i.e., 31% and 20% loss of inhibition, respectively). Interestingly, these losses of PLP-mediated inhibition could be rescued by covalent modification of each cysteine with MTSEA, a reagent that adds a lysine-like moiety (i.e. SCH2CH2NH3+) to the cysteine sulfhydryl group. Importantly, the replacement of non-binding site lysines (i.e., Lys-45, Lys-48, Lys-134, Lys-141) with cysteine resulted in little change in the PLP inhibition. Based upon these results, we conducted docking calculations with the CTP structural model leading to the development of a physical binding model for PLP. In combination, our data support the conclusion that PLP exerts its main inhibitory effect by binding to residues located within the two substrate binding sites of the CTP, with Lys-83 being the primary determinant of the total PLP effect since the replacement of this single lysine abolishes nearly all of the observed inhibition by PLP.

Probing the effect of transport inhibitors on the conformation of the mitochondrial citrate transport protein via a site-directed spin labeling approach

The present investigation utilized the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy to identify the effect of citrate, the natural ligand, and transport inhibitors on the conformation of the yeast mitochondrial citrate transport protein (CTP) reconstituted in liposomal vesicles. Spin label was placed at six different locations within the CTP in order to monitor conformational changes that occurred near each of the transporter’s two substrate binding sites, as well as at more distant domains within the CTP architecture. We observed that citrate caused little change in the EPR spectra. In contrast the transport inhibitors 1,2,3-benzenetricarboxylate (BTC), pyridoxal 5′-phosphate (PLP), and compound 792949 resulted in spectral changes that indicated a decrease in the flexibility of the attached spin label at each of the six locations tested. The rank order of the immobilizing effect was compound 792949 > PLP > BTC. The four spin-label locations that report on the CTP substrate binding sites displayed the greatest changes in the EPR spectra upon addition of inhibitor. Furthermore, we found that when compound 792949 was added vectorially (i.e., extra- and/or intra-liposomally), the immobilizing effect was mediated nearly exclusively by external reagent. In contrast, upon addition of PLP vectorially, the effect was mediated to a similar extent from both the external and the internal compartments. In combination our data indicate that: i) citrate binding to the CTP substrate binding sites does not alter side-chain and/or backbone mobility in a global manner and is consistent with our expectation that both in the absence and presence of substrate the CTP displays the flexibility required of a membrane transporter; and ii) binding of each of the transport inhibitors tested locked multiple CTP domains into more rigid conformations, thereby exhibiting long-range inter-domain conformational communication. The differential vectorial effects of compound 792949 and PLP are discussed in the context of the CTP homology-modeled structure and potential mechanistic molecular explanations are given.

Transport Inhibitors Cause Conformational Changes in the Yeast Mitochondrial Citrate Transport Protein Reconstituted in Liposomes as Demonstrated by EPR Spectroscopy

In order to directly observe conformational change in the mitochondrial citrate transport protein (CTP), we measured, in the presence and absence of inhibitors, the EPR spectra of spin-labeled single-Cys CTP mutants that were reconstituted in liposomes. We selected spin-label locations to report on substrate binding sites 1 and 2 (i.e., 187, 183, and 179), binding site 2 (39), TMDIII pointing away from the transport pathway (118), and a matrix-facing hydrophilic loop (47). In the absence of inhibitor, the EPR lineshapes show residue-dependent variations in mobility. Addition of external 1,2,3-benzenetricarboxylate (BTC), the defining inhibitor of the CTP, caused a modest, residue-dependent decrease in the mobile component and a concomitant increase in the immobile component. Addition of compound 792949, a novel, purely competitive inhibitor that we previously identified via high throughput in silico screening using the homology-modeled CTP in its cytosolic-facing conformation, yielded EPR spectra that contain a substantial increase in the immobile component at each location. We conclude that the two inhibitors cause CTP to assume different conformations, which vary significantly in their extent of immobilization.Surprisingly, in contrast to the large immobilizing effect observed upon the addition of extraliposomal inhibitor 792949, the inclusion of intraliposomal inhibitor caused only a minor spectral change. This observation indicates that binding of 792949 to CTP from the internal surface of the proteoliposomes (i.e., the matrix-facing conformation) occurs to a much lesser extent than does binding to CTP from the external surface (i.e., the cytosolic-facing conformation). We conclude that external 792949 affects spin-label mobility at both monomers within the functional homodimer suggesting a tight coordination of the two monomers. Supported by NIH grant GM-054642 to R.S.K.