The objective of the present investigation was to explore the effects of substrate and inhibitors on the conformation of the yeast mitochondrial CTP using the site-directed spin labeling approach of EPR spectroscopy. It is noteworthy that the EPR spectra observed at each of the six locations were consistent with the topological locations predicted by the CTP homology-modeled structure (Walters and Kaplan 2004
) and our proposed monomer-monomer interface in homodimeric CTP (Ma et al. 2005
), thereby providing new validation for these models. Furthermore, several key novel findings were obtained by our studies. First
, we demonstrate that citrate, the native substrate, does not cause any significant change in the spectra of spin label residing at multiple locations throughout the CTP, thus indicating that citrate binding to the CTP substrate binding sites does not alter the dynamics of the side-chains and the backbone of the CTP in the non-binding site regions of the transporter in a global manner. This finding is consistent with our expectation that in both the absence and presence of substrate the CTP displays the flexibility required of a membrane transport protein. Second
, each of the three inhibitors tested (), caused spectral changes that indicate varying degrees of immobilization of the spin label ( and ). 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
Experiments conducted with external
compound 792949 (, Panels B & C), a purely competitive inhibitor that is capable of spanning binding sites 1 and 2 (Aluvila et al. 2010
), yielded EPR spectra that indicate a concurrent substantial increase in the immobile component and decrease in the mobile component at each of the six locations tested, indicating that the effect is global. It is noteworthy that 792949 caused considerably greater immobilization than did BTC (compare Panel C in and ). These findings support the conclusion that the binding of compound 792949 causes a shift in the conformational equilibrium such that the side chain R1 in CTP becomes less mobile. The inhibitor-induced decrease in mobility could originate from increased tertiary interactions of R1 with nearby CTP domains and/or from a reduction in the CTP backbone motion (Columbus and Hubbell 2002
), either of which signifies a more locked, rigid CTP conformation. Relatedly, we note that as depicted in , an important difference between compound 792949 and either BTC or citrate is that the former has a length sufficient to span both substrate binding sites within the CTP, whereas BTC and citrate do not. We posit that it is these additional specific binding interactions of 792949 to both CTP substrate binding sites simultaneously (Aluvila et al. 2010
) that impose significantly more restriction on CTP mobility than observed with either BTC or citrate.
We also studied the effect of extra-liposomal PLP, a lysine-selective reagent that has been shown to inhibit the CTP, on the EPR spectrum. We observed that PLP causes significant immobilization of spin-label placed at six different locations within the CTP (). We note that the immobilizing effect by external PLP is less potent than observed with external 792949, but is more pronounced than observed with BTC or citrate. Consequently, we posit that external PLP causes an intermediate degree of locking of the CTP conformation, a finding that is consistent with our previous observation that PLP inhibits the CTP mainly via interactions of residue Lys83 in substrate binding site 1 and is of insufficient length to simultaneously span sites 1 and 2 in their entirety.
Our investigations into the vectorial
effect of the inhibitors compound 792949 and PLP on the lineshape of 179R1 ( and ) provide important clues regarding CTP function. For example, the finding that only upon addition of compound 792949 from the external
surface of the proteoliposomes do we observe substantial changes in the EPR spectra suggests that binding of 792949 to CTP from the internal
surface of the proteoliposomes occurs to a much lesser extent than does binding to the CTP from the external
surface. These data strongly support the conclusion that: i) in our proteoliposomal preparation the CTP is predominantly incorporated into the liposomal bilayer asymmetrically (i.e., unidirectionally; cytosolic-facing
conformation oriented outwards); and ii) in the cytosolic-facing
conformation of the CTP, substrate binding sites 1 and 2 are primarily accessible to the extra-liposomal surface. Further-more, it is noteworthy that previously we hypothesized, based on the facts that 792949 was identified via docking experiments using the cytosolic-facing
conformation of the CTP as the template and based on kinetic data, that 792949 binds exclusively to the cytosolic-facing
conformation of the CTP (Aluvila et al. 2010
). Our present findings on the vectorial effect of 792949 on EPR spectra provide additional support for this conclusion. Finally, if CTP functions as a homodimer (Kotaria et al. 1999
), our data with compound 792949 suggest that the conformational changes in each of the two monomers are tightly coordinated. Thus upon addition of 792949, the locking of one monomer’s conformation results in a similar locking of the other’s, thereby reducing spin-label mobility at both locations within the CTP and resulting in a highly immobilized spectrum.
Interestingly, we observed that intra-liposomal
PLP caused considerably more immobilization of the spin labels than did intra-liposomal
compound 792949. Moreover, in the case of PLP we observed a similar extent of immobilization when the inhibitor was added to either the intra
- or the extra-liposomal
compartments. Furthermore, the effect is approximately additive when PLP is added to both compartments. This is in sharp contrast to compound 792949, which exerts nearly its entire effect from the extra-liposomal
surface. These observations beg the question as to how PLP can interact with and immobilize the CTP from its intra-liposomal
surface, whereas compound 792949 cannot. We note several points. First
, as depicted in , in the cytosolic-facing
conformation of the CTP, which was used to identify compound 792949 via in silico
docking, the translocation pathway may be closed near the matrix surface (denoted as a dotted line). Thus, it is not surprising that the inhibitor is unable to bind to the cytosolic-facing
conformation from its internal surface. Furthermore, the minimal change in the EPR spectrum caused by intra-liposomal
792949 suggests that this inhibitor does not bind significantly to the other CTP monomer that exists in the matrix-facing
conformation within the homodimer. Second
, with regard to PLP we note that the CTP contains numerous lysines both near the matrix surface as well as within the translocation pathway. Furthermore, we previously demonstrated that most (i.e., 78%) of the inhibition of transport caused by extra-liposomal
PLP arises from its binding to Lys-83 in binding site 1, whereas the remaining inhibition originates from PLP binding to site 2 residues Lys-37 and/or Lys-239. Presumably, the immobilization of the EPR spectra caused by external PLP is mediated via binding to these same residues. The question then arises as to the mechanism by which intra-liposomal
PLP causes the observed spectral changes. We note that in the CTP homodimer the cytosolic-facing
and the matrix-facing
conformations will always exist in equal measure. As depicted in , we postulate that in the cytosolic-facing
conformation, binding site 1 is accessible to the external aqueous milieu but not the internal aqueous compartment. Furthermore, we propose that in the matrix-facing
conformation, CTP binding site 1 exists in a different conformation that may or may not remain accessible to the external
milieu (Aluvila et al. 2010
), as indicated by the dotted line in the figure and is not extensively accessible to internal
PLP. Thus, we propose that the intra-liposomal
PLP effect is due either to a more pronounced effect on binding site 2 lysines in this matrix-facing
conformation and/or to the binding of PLP to other internal lysines. With respect to the latter idea, we note that conserved lysines exist within CTP domains that are located near the internal surface of the bilayer. We postulate that these residues may participate in the formation of a third substrate binding site in the matrix-facing
conformation of the CTP and that the additional immobilization caused by intra-liposomal
PLP may arise via binding to this hypothetical third site. Finally, we note that at the concentrations tested (i.e., 15 mM PLP and 10 mM compound 792949), the total extent of immobilization achieved in the presence of intra
- plus extra-liposomal
inhibitor is similar for both PLP and compound 792949. Significantly, the ability of these two inhibitors to lock domains within the CTP to different extents depending on the site of their addition, will provide complementary, yet overlapping means for probing CTP architecture.
Fig. 8 Schematic representation of substrate binding sites within homodimeric CTP located within the mitochondrial inner membrane bilayer. Binding sites 1 and 2 and a hypothetical binding site 3 are depicted in the cytosolic-facing and matrix-facing conformations (more ...)
Our findings with spin label placed at six different locations within the CTP indicate that inhibitor binding causes global conformational changes that result in the immobilization of the spin label at each location tested. Moreover, we note that, with each of the three inhibitors tested, based on RMS values, we see the same pattern of residue-dependent extent of immobilization (i.e., 183R1 > 187R1 >179R1 > 39R1 > 47R1 > 118R1). For example, with a given ligand, TMD IV residues 179R1, 183R1, and 187R1, which are located on the same face of this helix, all exhibit greater extents of immobilization than are observed at the other locations tested. This result strongly suggests that immobilization of these spin labels is due to a rigid body motion of the TMD IV helix which arises from inhibitor binding to CTP substrate binding sites 1 and/or 2. One can envision that this motion might be either a twisting of the helix that results in the placement of the TMD IV spin labels near the surface of an adjacent helix and/or by translation of the TMD IV helix toward the central axis of the CTP transport pathway thereby causing a decrease in the distance between CTP TMD helices forming new spin label side chain interactions that restrict motion. With respect to 39R1 we note that it is located near the matrix end of TMD I, likely points towards the matrix compartment, and is in close proximity to binding site 2 and thus reports on conformational changes at this site. Similar to most of the other residues examined, its EPR spectra exhibited an intermediate mobility in the absence of inhibitor ( and ). As observed with the other residues, binding of externally added inhibitor results in a large increase in the immobile population and a concurrent decrease in the mobile population of spin label at this location. Thus, this domain also becomes less flexible for the reasons described above. Finally, we note that the four locations (i.e., 39, 179, 183, 187) that report most directly on substrate binding sites 1 and/or 2, display the greatest changes in the EPR spectrum upon addition of inhibitor.
Two additional locations were studied (i.e. 47R1 and 118R1) which are not located near substrate binding sites 1 or 2. We note that 47R1 is located in Matrix Loop A which connects TMDs I and II (see ), and displays a mobility comparable to that of R1 located on TMD IV (ie., at locations 179, 183, and 187) in the absence of inhibitor. This finding suggests that either this loop is partially structured and/or unlike the depiction in , it may in fact have substantive tertiary interactions with other nearby domains that limit its mobility. In the presence of inhibitors, its mobility also is reduced suggesting long-range conformational communication between the inhibitor binding sites and this domain.
Previously, based on both kinetic analysis and molecular modeling (Ma et al. 2005
) we hypothesized that residue 118 resides near the monomer-monomer interface in the homodimer and may be involved in coordinating the conformational change between the two monomers. Interestingly, we note that despite the fact that the side chain of residue 118 points away from the CTP monomer in the direction of the lipid bilayer, 118R1 nonetheless displays a very immobilized EPR spectra, indicating considerable tertiary contact at this location. In the presence of CTP inhibitors, its mobility is further diminished () supporting the notion that conformational communication may occur between the substrate binding sites and the monomer-monomer interface in homodimeric CTP.
Relatedly, the following note of caution is in order. The molecular interpretations that we have posited to account for the observed EPR data are based in part on our homology-modeled structure of the CTP (Walters and Kaplan 2004
) which, as mentioned above, was developed from the crystallographic structure of the mitochondrial ADP/ATP carrier (Pebay-Peyroula et al. 2003
). Consequently, the validity of these interpretations relies upon the accuracy of the CTP homology model. We have a high degree of confidence in the correctness of this model since it has accurately predicted the composition of the CTP substrate translocation pathway and the substrate binding sites, as well as a specific steric interaction between Gln182 in TMD IV and Leu120 in TMD III, all of which have been experimentally verified (Kaplan et al. 2000a
; Ma et al. 2004
). Nonetheless, a high-resolution 3-dimensional crystal structure of the CTP will be needed to unequivocally confirm the validity of certain of the molecular explanations posited for the present findings.
Finally, a critical conclusion derived from these experiments, is that at each of the six sites examined, which reside in vastly different CTP domains, inhibitor binding results in long-range conformational changes as evidenced by changes in EPR spectra. These results support the hypothesis that transport of CTP substrates occurs via a long-range coordinated motion between domains within this carrier. Our future experiments will be directed toward understanding the nature of these conformational changes and the composition of the monomer-monomer interface by directly measuring distances between CTP domains, in the presence and absence of various ligands. Furthermore, the discovery of inhibitors that lock CTP into an immobilized conformation(s), which may represent one or more of the conformations that CTP assumes during its transport cycle, may provide key tools in the search for conditions that yield well-diffracting CTP crystals and thereby pave the way for an atomic resolution structure of the CTP.