Perspectives in Pharmacology Structures and Models of Transporter Proteins

Transporter proteins in biological membranes may be divided into channels and carriers. Channels function as selective pores that open in response to a chemical or electrophysiological stimulus, allowing movement of a solute down an electrochemical gradient. Active carrier proteins use an energy producing process to translocate a substrate against a concentration gradient. Secondary active transporters use the movement of a solute down a concentration gradient to drive the translocation of another substrate across a membrane. ATP-binding cassette (ABC) transporters couple hydrolysis of adenosine triphosphate (ATP) to the translocation of various substrates across cell membranes. High-resolution three-dimensional structures have now been reported from X-ray crystallographic studies of six different transporters, including two ATP-binding cassette (ABC) transporters. These structures have explained the results from many previous biochemical and biological studies and shed new light on their functional mechanisms. All these transporters have -helical structures of the membrane-spanning domains, as suggested from many previous studies, and some of the helices have irregular shapes with kinks and bends. Together these crystal structures demonstrate the large flexibility of transporter proteins and that substantial movements take place during the substrate translocation process, which to a certain extent may distinguish active carriers from channel proteins. These structures and other low-resolution structures of membrane proteins have served as a basis for construction of three-dimensional protein models that have provided insight into functional mechanisms and molecular structures and enabled formulation of new hypotheses regarding transporter structure and function, which may be experimentally validated. Twelve years ago a review article described the growth of information on transporters as a “transporter explosion” (Uhl and Hartig, 1992). Since then, genome sequencing has made information on transporters expand in magnitude at an even more rapid rate, and the amount of information on their structures and functional mechanisms seems to have grown exponentially. A transporter protein classification system (TC) proposed in 1999 was based on phylogenetic analysis and functional properties of about 250 families of sequencerelated transporters identified in bacteria, archaea, and eukaryotes (Saier, 1999). Three years later, nearly 400 families were included in the TC classification system, which then had been adopted by the International Union of Biochemistry and Molecular Biology (Busch and Saier, 2002). Phylogenetic analysis of integral membrane transport protein sequences has shown that families of transport proteins of similar topology have evolved independently of each other, at different times in evolutionary history, using different routes (Saier, 1999, 2000; Busch and Saier, 2002). Now that the detailed three-dimensional molecular structures of some transporter proteins have been reported, the possible similarity in protein-folding patterns between these and other transporters is of crucial importance. Similar folding patterns between any protein and one for which the crystal structure is known would enable construction of a fairly The present study was supported by Grant 136158/310 from the Norwegian Research Council (NFR). Article, publication date, and citation information can be found at http://jpet.aspetjournals. org. DOI: 10.1124/jpet.103. 059972. ABBREVIATIONS. TC, transporter protein classification system; MFS, major facilitator superfamily; 5-HTT, serotonin transporter; ABC, ATPbinding cassette; AcrA, membrane fusion protein; AcrB, resistance-nodulation-cell division transporter; DAT, dopamine transporter; ECM, electron cryo microscopy; EmrE, bacterial multidrug transporter; GlcV, ATP-binding cassette-ATPase subunit of glucose transporter; GlpT, glycerol-3phosphate transporter; LacY, lactose permease; MDR, multidrug resistance; MsbA, lipid flippase; NBD, nucleotide-binding domain; NET, norepinephrine transporter; NhaA, Na /H antiporter; OxlT, oxalate transporter; P-site, auto-phosphorylation site; P-gp, P-glycoprotein; RND, resistance-nodulation cell division; SERCA1a, skeletal muscle sarcoplasmic reticulum calcium ATPase; TMH, transmembrane -helix; TolC, outer membrane channel of multidrug exporter system. 0022-3565/04/3093-853–860$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 309, No. 3 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 59972/1150468 JPET 309:853–860, 2004 Printed in U.S.A. 853 at A PE T Jornals on July 9, 2017 jpet.asjournals. org D ow nladed from accurate three-dimensional protein model of the unknown structure using the related crystal structure as a template and modern computational techniques. However, although atomic resolution crystal structures of soluble proteins have been reported in an amazingly increasing number, such progress has not been made in terms of transporters and other membrane proteins, which have proven extremely difficult to crystallize for two main reasons. One is related to the amphipathic nature of their surface, with a hydrophobic area in contact with membrane phospholipids and polar surface areas in contact with the aqueous phases on both sides of the membrane (Ostermeier and Michel, 1997). Second, the majority of medically important membrane proteins are present in tissues at very low concentrations, making overexpression a prerequisite for structural studies, and this often has proven to be very difficult. In spite of recent advances in overexpression technology, bacterial membrane proteins still are the most amenable for structure determination, simply because they often can be easily expressed in large quantities (Tate, 2001). As shown in Table 1, five of the six currently reported high-resolution transporter structures are from Escherichia coli. Their inherent conformational flexibility may also contribute to the difficulties in obtaining stable crystals of transport proteins. Using inhibitors to rigidify their structure has therefore been proposed as a possible strategy for crystallization of flexible receptors and transporters (Tate, 2001). Lactose permease was only crystallized in a mutant form (Abramson et al., 2003), which shows greater thermal stability that the wild type and markedly reduced ligand-induced conformational changes (Smirnova and Kaback, 2003). The experimental obstacles involved in crystallization and structure determination of membrane proteins are illustrated by the fact that before determining the crystal structure of a lipid flippase (MsbA) from E. coli, the group cloned, overexpressed, and purified more than 20 different bacterial ABC transporters, and tested 96,000 different crystallization conditions for several multidrug resistance (MDR)-ABC transporters using about 20 different detergents (Chang and Roth, 2001). Classification of Transport Proteins In the TC classification system, transporters are divided into two main classes: channels and carriers (Saier, 2000; Busch and Saier, 2002), as indicated in Fig. 1. Channel proteins function as selective pores that open in response to a chemical or electrophysiological stimulus, allowing movement of a solute down an electrochemical gradient. Active carrier proteins translocate substrates across a biological membrane against a concentration gradient. To do this, the transporter must couple the carrier process to another energy-producing process and also be able to prevent the two processes from occurring uncoupled in their energetically favorable directions. Secondary active transporters use the movement of a solute down a concentration gradient to drive the translocation of an other substrate across a membrane against a concentration gradient, by symport or antiport mechanisms. Symporters (cotransporters) catalyze the transport of two or more molecular species in the same direction, and antiporters (exchange transporters) catalyze the exchange of one or more T B L E 1 T h re edi m en si on al tr an sp or te r st ru ct u re s P ro te in O rg an is m T ra n sm em br an e do m ai n s O th er D om ai n s S tr u ct u re D et er m in at io n R ef er en ce