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

Abstract

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. SCH 2 CH 2 NH 3 + ) 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

“…Thus inhibitor binding appears to reduce CTP flexibility, perhaps locking it into one of the conformation(s) that is normally assumed during the transport cycle. The latter point is supported by our earlier findings that each of the inhibitors tested display a strong competitive component in their inhibition mechanism (Remani et al 2008; Aluvila et al 2010). …”

“…Finally, PLP is a lysine-selective covalent modification reagent which in the absence of reducing agent forms a reversible Schiff base (Lundblad 1991). We have previously demonstrated that PLP behaves as a linear mixed inhibitor of the CTP, with a predominantly competitive component and displays a K ic of 3.6±0.8 mM (Remani et al 2008). PLP exerts its main inhibitory effect via binding to Lys-83 in binding site 1, and a small effect via binding to residues Lys-37 and Lys-239 in site 2.…”

“…Interpretation of chemical modification ± substrate protection data obtained with site specific mutants (Kaplan et al 2000a; Ma et al 2004, 2005), within the framework of the CTP homology model has resulted in the identification of substantial portions of the substrate translocation pathway. Moreover, kinetic analysis of single-Cys CTP mutants in combination with molecular modeling using our homology-modeled structure has: i) resulted in the identification of two substrate binding sites per CTP monomer that reside at increasing depths within the bilayer (Ma et al 2007); and ii) enabled characterization of the inhibition mechanism of BTC, the classical inhibitor of the CTP, as well as of PLP, a lysine-selective reagent (Remani et al 2008). Recently, in silico screening of the ZINC database of commercially available compounds, followed by experimental testing of selected compounds, led to the discovery of the first purely competitive inhibitor of the CTP (i.e., compound 792949) (Aluvila et al 2010).…”

Warburg’s Ghost—Cancer’s Self-Sustaining Phenotype: The Aberrant Carbon Flux in Cholesterol-Enriched Tumor Mitochondria via Deregulated Cholesterogenesis

“…Of the more than 50 mitochondrial membrane substrate/ion transporters of the SLC25 superfamily that have been identified and characterized to date (for reviews see: Kaplan, 2001;Palmieri and Monné, 2016), Kaplan's laboratory (Remani et al, 2008;Aluvila et al, 2010;Sun et al, 2010;Nie, 2017) has provided significantly detailed information on the structure, substrate transport mechanism and analysis of inhibition by BTC and other potential inhibitors of the citrate-malate CTP carrier, as well as on the PMCT membrane transporter. PMCT is the more recently characterized plasma membrane citrate transporter variant in humans and has garnered considerable attention since it serves as an additional (and, so far only) channel for the influx of citrate into the cytosol from the extracellular space (Mycielska et al, 2009).…”

Towards closing the remaining gaps in photorespiration – the essential but unexplored role of transport proteins

“…Neither group examined the transport of PLP. Due to the fact that PLP reacts very easily with the amino acid lysine, forming a Schiff's base with the epsilon amino group, PLP is often used in transport assays as an effective inhibitor (Remani et al 2008). Hence, it seems unlikely that Vitamin B 6 is transported in this form, but rather as pyridoxamine or pyridoxine.…”