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

Abstract

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.