might change in the future if more research and technologies are applied to this area.

5.3 Structural Models of Transport Proteins and Methods to Design Substrates

The primary and, to a lesser extent, secondary structures of many transport systems are known. Because of the absence of suitable crystallization methods, however, only a few polytopic membrane proteins have yielded to X-ray crystallographic analyses (127-130), resulting in high resolution three-dimensional structural information. Recently, combinatorial approaches have been applied to successfully crystallize membrane proteins in a high throughput approach, resulting in the structure of a homolog of the multidrug-resistant ATP-binding cassette transporters to a resolution of 4.5 A (131). In this study, approximately 96,000 crystallization conditions using about 20 detergents were tested to yield crystals with good quality for X-ray structure determination. Unfortunately, the resulting protein was not functionally active, but the structural information corroborated several previously obtained experimental results. The combinatorial crystallization approach used by Chang and Roth (131) will likely stimulate other researchers to attempt crystallization of other important membrane proteins.

Meanwhile, we base our views of solute transport on molecular models that provide working models of transport systems (132-134). It is well recognized that any two proteins that show sequence homology (i.e., share sufficient primary structural similarity to have evolved from a common ancestor) will prove to exhibit strikingly similar three-dimensional structures (135). Furthermore, the degree of tertiary structural similarity correlates well with the degree of primary structural similarity. Phylogenetic analyses allow application of modeling techniques to a large number of related proteins and additionally allow reliable extrapolation from one protein family member of known structure to others of unknown structure. Thus, once three-dimesnional structural data are available for any one family member, these data can be applied to all other members within limits dictated by their degrees of sequence similarity.

5.4 Techniques for Studying Integral Membrane Protein Structure

There are various approaches to study the topography of integral membrane proteins in the absence of a resolved crystal structure. In vitro and in vivo translation of constructs containing one or more transmembrane sequences, epitope localization, reaction with sided reagents, and proteolysis of the purified membrane-inserted protein have all been used individually or in combination. (Table 8.3). Each has advantages and disadvantages, and only rarely does one method enable conclusive topographic analysis of the membrane-embedded segments of a polytopic integral membrane protein.

Considerable progress has been made over the past 10 years by the group of Kaback at UCLA, who have taken numerous biophysical approaches toward studying the structure of the bacterial transporter lactose permease (136). Table 8.3 lists the techniques that this and other groups have used to solve the structure and topology of membrane transporters in the absence of a crystal structure.

5.4.1 Membrane Insertion Scanning. Membrane topology of transporter proteins can be

Table 8.3 Techniques for Studying Polytopic Membrane Protein Structure

1. Site-directed Ala scanning

2. Membrane insertion scanning

3. Site-directed thiol crosslinkers

4. Excimer fluorescence

5. Engineered divalent metal-bindingsites

7. Metal-spin-labeling interactions

8. Site-directed chemical cleavage

9. Identification of discontinuous monoclonal antibody probes

10. iV-glycosylation site engineering predicted by various computer algorithms; however, none of the existing methods can definitively define these regions, often producing a "sliding window" of groups of hydrophobic amino acid residues that are suitable candidates for the formation of a membrane-spanning segment. Examples of these software programs are TopPred II (137) (http://www., the PHD Topography neural network system developed by Rost and coworkers (138) (http://www.embl-heidelberg. de/predictprotein), or the hidden Markov model recently described by Tusnady and Simon (139) ( Sachs and colleagues at UCLA pioneered a technique aptly named "membrane insertion scanning" to determine whether a sequence of amino acids is capable of spanning a membrane. They tested the ability of individual protein segments to function as signal anchors for membrane insertion by placement between a cytoplasmic anchor encompassing the first 101 amino acids of the rabbit H+,K+-

subunit and a glycosylation flag sequence consisting of five N-linked glyco-sylation sites located in the C-terminal 177 amino acids of the rabbit HK MD subunit (140).Stop transfer properties of hydrophobic sequences can be examined in a similar manner using the first 139 N-terminal amino acids of the rabbit H+,K+-ATPase subunit (HKM1) containing the first membrane sequence of the H+,K+-ATPase as a signal anchor upstream of individual predicted transport transmembrane sequences linked to the N-glycosylation flag. The membrane insertion scanning technique has been used to determine the membrane topology of various transporters, chan-

| nels, and receptors. A common criticism cf I membrane insertion studies is the placement | of a hydrophobic amino acid sequence out of | its physiological or environmental context, | thus "forcing" an abbreviated sequence | through the membrane. Transmembrane re-| gions in polytopic membrane proteins may rei quire flanking topogenic information to become integrated into the lipid bilayer and, thus, topology determination by this technique alone may not be definitive.

5.4.2 Cys-Scanning Mutagenesis. Studying the lactose permease of Escherichia coli, a

[ polytopic membrane transport protein that catalyzes j3-galactoside/H+ symport, Frill-ingos and colleagues (141) used Cys-scanning mutagenesis to determine which residues play an obligatory role in the mechanism and to create a library of mutants with a single-Cys residue at each position of the molecule for structure/function studies. In general, this type of study will define amino acid side chains that play an irreplaceable role in the transport mechanism and positions where the reactivity of the Cys replacement is altered upon ligand binding. Furthermore, helix packing, helix tilt, and ligand-induced conformational changes can be determined by using the library of mutants in conjunction with a battery of site-directed techniques.

5.4.3 N-Glycosylation and Epitope Scanning Mutagenesis. A technique that can be used in addition to membrane insertion scanning is based on the fact that N-glycosylation occurs only on the luminal side of the endoplasmic reticulum. This method has been successfully used to determine which domains of the receptors/channels are located extracellularly. In general, a glycosylation-free mutant is first designed and subsequently, N-glycosylation consensus sequences (NXS/T) are engineered into hydrophilic regions of an aglycomutant. Based on the positioning of the glycosylation groups within the extracellular membrane, a molecular weight shift may indicate successful glyco-sylation and reveals definitive information on protein topology. A drawback of this technique is the dependency of glycosylation on efficiency and accessibility of the concensus sequence to the glycosylation machinery;

thus, nonglycosylated mutants do not provide conclusive information about the topology and these data should be interpreted carefully. Regardless, the topology of various proteins has been successfully solved using this technique, including the sodium-dependent glucose transporter (SGLT1) (142) and the 7-ami-nobutyric acid transporter GAT-1 (143).

Alternatively, small peptide epitopes can be inserted into the extramembranous parts of an SLC prote in that are recognized by a well-characterized monoclonal antibody. Covitz and colleagues (144) used this technique to determine the topology of the peptide transporter, PepTl. An epitope tag, EYMPME, was inserted into different extramembranous locations of hPEPTl by site-directed mutagenesis. The membrane topology was solved by labeling reconstituted, functionally active, EYMPME-tagged hPEPTl mutants with an anti-EYMPME monoclonal antibody in non-permeabilized and permeabilized cells.

5.4.4 Excimer Fluorescence. Site-directed excimer fluorescence (SDEF) and site-directed spin labeling (SDSL) are two particularly useful techniques to study proximity relationships in membrane helices. The experiments are based on site-directed pyrene labeling of combinations of paired Cys replacements in a mutant devoid of Cys residues. Because pyrene exhibits excimer fluorescence if two molecules are within about 3.5 Â, the proximity between paired labeled residues can be determined. Moreover, interspin distances in the range of 8-25 Â between two spin-labeled Cys residues can be measured in the frozen state. Using this technique, Kaback and coworkers showed that ligands of the lac per-mease cause a dramatic increase in reactivity that is consistent with the notion that the mutated amino acid positions are transferred into a more hydrophobic environment (145,146).

5.4.5 Site-Directed Chemical Cleavage. The insertion of short reporter sequences (e.g., factor Xa protease cleavage sites) into hydrophilic loops has proved to be a useful alternative to N-glycosylation scanning mutagenesis. However, this approach requires isolation of homogeneous preparations of intact membranes and many tedious control experiments, and experimental difficulties associated with protease accessibility are well documented (147).In general, in-frame factor Xa protease sites are inserted into a target sequence at positions within the NH2- and COOH-terminal domains, and into hydrophilic loops. The factor Xa protease recognizes the tetrapeptide motif IEGR and specifically cleaves the protein sequence COOH-terminal of the arginine residue (148).Generally, the recognition motif is tandomly (IEGRIEGR) inserted to increase the probability of cleavage (149). After digestion of purified protein vesicles with the factor Xa enzyme, fragments are isolated on SDS-PAGE and can be analyzed to further determine membrane topology.

5.5 Case Studies

5.5.1 P-Clycoprotein: Understanding the Defining Features of Regulators, Substrates, and Inhibitors. The ABC efflux transporter P-gly-coprotein (P-gp) is a large 12 transmembranedomain bound protein initially noted to be present in certain malignant cells associated with the multidrug resistance (MDR) phenomenon that results from the P-gp-mediated active transport of anticancer drugs from the intracellular to the extracellular compartment (150). However, P-gp is normally expressed at many physiological barriers including the intestinal epithelium, canalicular domain of hepatocytes, brush border of proximal tubule cells, and capillary endothelial cells in the central nervous system (CNS) (150) .Expression of P-gp in such locations results in reduced oral drug absorption and enhanced renal and biliary excretion of substrate drugs (151).Moreover, P-gp expression at the blood-brain barrier is a key factor in the limited CNS entry of many drugs. The expressed level of P-gp as well as altered functional activity of the protein attributed to genetic variability in the MDR1 gene also appears to impact the ability of this transporter to influence the disposition of drug substrates (152).

Interestingly, cytochrome P450 3A4 a drug-metabolizing enzyme with broad substrate specificity, appears to coexist with P-gp in organs such as the intestine and liver. These observations led to the hypothesis that there may be a relationship between these two proteins in the drug disposition process. Wacher et al. have described the overlapping substrate specificity and tissue distribution of CYP3A4 (153) and P-gp. Schuetz et al. found that modulators and substrates coordi-nately upregulate both proteins in human cell lines (154). Siniilarly, P-gp-mediated transport was found to be important in influencing the extent of CYP3A induction in the same cell lines and also in mice (155). More recent data have suggested that there may be a dissociation of inhibitory potencies for molecules against these proteins. Although some molecules can interact with CYP3A4 and P-gp to a similar extent, for the most part the potency of inhibition for CYP3A4 did not predict the potency of inhibition for P-gp, and vice versa (58) .Moreover, not all CYP3A substrates such as midazolam and nifedipine are P-gp substrates (156).The key to this linkage between P-gp and CYP3A4 appears to be their coregu-lation by the pregnane-X-receptor at the level of transcription (157, 158). Recently, with the description of the X-ray crystal structure of this protein with SR12813 bound, we may be closer to understanding the structural features necessary for PXR ligands. In addition, a pharmacophore for PXR ligands may also enable us to select new drugs that are less likely to induce P-gp and CYP3A4 (159).

To account for the observed broad substrate specificities for both CYP3A4 and P-gp, the presence of multiple drug binding sites has been proposed (160-163). The first elegant experimentally determined signs of a complex behavior for P-gp appeared in 1996 when cooperative, competitive, and noncompetitive interactions between modulators were found to interact with at least two binding sites in P-gp (164). The multiple site hypothesis was confirmed by other groups (165-167). Subsequent results have indicated there may be three or more binding sites (168).Steady-state kinetic analyses of P-gp-mediated ATPase activity using different substrates indicate that these sites can show mixed-type or noncom-petitive inhibition indicative of overlapping substrate specificities (169). Other researchers have determined that immobilized P-gp demonstrates competitive behavior between vinblastine and doxorubicin, cooperative allosteric interactions between cyclosporin and vinblastine or ATP, and anticooperative allosteric interactions between ATP, vinblastine, and verapamil (170). Clearly allosteric behavior by multiple substrates, inhibitors, or modulators of CYP3A4 or P-gp complicates predicting the behavior and drug-drug interactions of new molecules in vivo and has important implications for drug discovery.

In terms of understanding P-gp structure-activity relationships, photoaffinity experiments have been valuable in defining the cyclosporin binding site in hamster P-gp (171) and indicating that trimethoxybenzoylyohim-bine (TMBY) and verapamil bind to a single or overlapping sites in a human leukemic cell line (172). Additional studies have shown that TMBY is a competitive inhibitor of vinblastine binding to P-gp (173). The P-gp modulator LY 335979 has been shown to competitively block vinblastine binding (173), whereas vinblastine itself can competitively inhibit verapamil stimulation of P-gp-ATPase (172). With the growth in knowledge derived from these and other studies, it would be valuable to use structural information to define whether unrelated molecules are likely to interact with

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