Mapping Conformational Heterogeneity of Mitochondrial Nucleotide Transporter in Uninhibited States

One of the less understood aspects of membrane transporters is the dynamic coupling between conformational change and substrate transport, because the dynamic uninhibited states of transporters have been largely inaccessible for structural studies. Here, we use NMR approaches to investigate conformational heterogeneity of the GTP/GDP carrier (GGC) from yeast mitochondria. NMR residual dipolar coupling (RDC) analysis of GGC in DNA-origami nanotube liquid crystal shows that several structured segments have different generalized degree of order (GDO), indicating the presence of conformational heterogeneity. Complete GDO mapping reveals asymmetry between domains of the transporter and even within certain transmembrane helices. Substrate titration measurements show that nucleotide binding partially reduces local structural heterogeneity, and that the substrate binds to multiple sites along the transport cavity. Our observations suggest that mitochondrial carriers in the uninhibited states are intrinsically plastic and structural plasticity is asymmetrically distributed among the three homologous domains.