J Membrane Biol (2010) 234:113–123 DOI 10.1007/s00232-010-9239-4 O R I G I N A L A R T I C L E S Tryptophan Scanning Analysis of the Membrane Domain of CTR-Copper Transporters Christopher J. De Feo • Sara Mootien • Vinzenz M. Unger Received: 4 November 2009 / Accepted: 19 February 2010 / Published online: 12 March 2010 The Author(s) 2010. This article is published with open access at Springerlink.com Abstract Membrane proteins of the CTR family mediate cellular copper uptake in all eukaryotic cells and have been shown to participate in uptake of platinum-based antican- cer drugs. Despite their importance for life and the clinical treatment of malignancies, directed biochemical studies of CTR proteins have been difﬁcult because high-resolution structural information is missing. Building on our recent 7A˚ structure of the human copper transporter hCTR1, we present the results of an extensive tryptophan-scanning analysis of hCTR1 and its distant relative, yeast CTR3. The comparative analysis supports our previous assignment of the transmembrane helices and shows that most function- ally and structurally important residues are clustered around the threefold axis of CTR trimers or engage in helix packing interactions. The scan also identiﬁed residues that may play roles in interactions between CTR trimers and suggested that the ﬁrst transmembrane helix serves as an adaptor that allows evolutionarily diverse CTRs to adopt the same overall structure. Together with previous bio- chemical and biophysical data, the results of the tryptophan C. J. De Feo S. Mootien V. M. Unger (&) Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06510, USA e-mail: Vinzenz.Unger@yale.edu Present Address: S. Mootien L2 Diagnostic, New Haven, CT 06511, USA scan are consistent with a mechanistic model in which copper transport occurs along the center of the trimer. Keywords Structure and function of transport proteins Protein biochemistry Molecular structure of membrane transporters Membrane transport Biophysical techniques in membrane research Introduction Fundamentally important processes such as oxygen reduction, detoxiﬁcation of radicals, the synthesis of con- nective tissue and neurotransmitters rely on the redox properties of the essential trace metal copper. In excess, however, copper is toxic to the cell. To avoid the harmful effects of copper ions while ensuring their speciﬁc delivery to metalloenzymes that require copper, cells employ a sophisticated homeostatic network whose core components are membrane-bound transporters, intracellular chaperones and metal-sensitive transcriptional regulators (Cobine et al. 2006; Huffman and O’Halloran 2001; Kim et al. 2008; Lutsenko et al. 2007). Malfunction and imbalances in this complex network can be fatal. For instance, Menkes and Wilson diseases, genetic disorders characterized by the accumulation and depletion of copper in certain tissues, are caused by mutations in two copper-transporting ATPases (DiDonato and Sarkar 1997). Moreover, aberrations in copper metabolism contribute to the etiology of neurode- generative diseases including Alzheimer and Creutzfeldt- Jakob diseases (Gaggelli et al. 2006; Macreadie 2008; Millhauser 2007). The clinical relevance and contributions of copper ions to fundamental biological processes provide strong incentives to understand cellular copper homeostasis at the molecular level. 123