Membrane transport proteins structural models

Relatively few membrane transport proteins have been structurally characterized. Some of the best understood examples to date are the lactose permease and glycerol-3-phosphate transporter and the Ca + P-type ATPase (which is a primary ion pump). Other structurally well-characterized transport proteins include the bacterial porins and siderophore receptor proteins. In addition structures have been determined for several ion channels and additional bacterial transporters that are either directly relevant to or models for proteins important in drug transport. The following web sites maintained by Hartmut Michel and Stephen White respectively, contain exceptionally useful listings of these and other solved membrane protein structures and are frequently updated ... 

In the rat, the observed effects of oral LD50 reflect both the intrinsic toxicity at the ultimate biophase site of action and the factors influencing distribution, membrane transport, protein binding, metabolism and excretion. The manifestation of acute mammalian toxicity is hence a much more complex response than can be described with the use of log alone, although individual processes such as bioavailability and adsorption depend on lipophilic-ity. The fit to a common QSAR model requires that each of these processes has similar structural dependences, qualitatively and quantitatively, within a given class of compounds. If a different process becomes predominant (i.e. rate limiting), the structure-toxicity relationship must alter thus the compound will be an outlier even when the principal mechanism of intrinsic toxicity remains the same. 

An additional feature in Fig. 2 worth noting is the amino-terminal 160 amino acids of mercuric reductase that lacked a fixed position in the crystal and therefore were not part of the solved structure. These contain the sequence that is homologous to MerP and postulated to be a mercurybinding domain. This region is drawn in Fig. 2 as an extension from the protein perhaps it functions like a baseball mitt that catches Hg from the membrane transport proteins and delivers Hg " to the carboxyl-terminal catalytic binding site, so that, as in the bucket brigade model above, Hg " is never found free within the cell. Mutant strains with the transport system but lacking the MerA detoxification enzyme are hypersensitive to mercury salts, as they accumulate Hg " but cannot get rid of it. After reduction by NADH (via FAD and the active site cysteine pair), metallic Hg is released... 

FIGURE 10.18 A model for the structure of the a-factor transport protein in the yeast plasma membrane. Gene duplication has yielded a protein with two identical halves, each half containing six transmembrane helical segments and an ATP-binding site. Like the yeast a-factor transporter, the multidrug transporter is postulated to have 12 transmembrane helices and 2 ATP-binding sites. 

All of the transport systems examined thus far are relatively large proteins. Several small molecule toxins produced by microorganisms facilitate ion transport across membranes. Due to their relative simplicity, these molecules, the lonophore antibiotics, represent paradigms of the mobile carrier and pore or charmel models for membrane transport. Mobile carriers are molecules that form complexes with particular ions and diffuse freely across a lipid membrane (Figure 10.38). Pores or channels, on the other hand, adopt a fixed orientation in a membrane, creating a hole that permits the transmembrane movement of ions. These pores or channels may be formed from monomeric or (more often) multimeric structures in the membrane. 

Poor intestinal absorption of a potential drug molecule can be related to poor physicochemical properties and/or poor membrane permeation. Poor membrane permeation could be due to low paracellular or transcellular permeability or the net result of efflux from transporter proteins including MDRl (P-gp) or MRP proteins situated in the intestinal membrane. Cell lines with only one single efflux transporter are currently engineered for in vitro permeability assays to get suitable data for reliable QSAR models. In addition, efforts to gain deeper insight into P-gp and ABC on a structural basis are going on [131, 132]. 

Balaz and Lukacova (1999) attempted to model the partitioning of 36 non-ionizable compounds in 7 tissues. Amphiphilic compounds, or those possessing extreme log Kow values, tended to show complex distribution kinetics because of their slow membrane transport. However for the non-amphiphilic, non-ionizable compounds with non-extreme log Kow values studied it should be possible to characterize their distribution characteristics based on tissue blood PCs. Distribution is dependent on membrane accumulation, protein binding, and distribution in the aqueous phase. As these features are global rather than dependent on specific 3D structure, distribution is not expected to be structure-specific. In this study, tissue compositions in terms of their protein, lipid, and water content were taken from published data. This information was used to generate models indicating that partitioning was a non-linear function of the compound s lipophilicity and the specific tissue composition. 

The percolation model suggests that it may not be necessary to have a rigid geometry and definite pathway for conduction, as implied by the proton-wire model of membrane transport (Nagle and Mille, 1981). For proton pumps the fluctuating random percolation networks would serve for diffusion of the ion across the water-poor protein surface, to where the active site would apply a vectorial kick. In this view the special nonrandom structure of the active site would be limited in size to a dimension commensurate with that found for active sites of proteins such as enzymes. Control is possible conduction could be switched on or off by the addition or subtraction of a few elements, shifting the fractional occupancy up or down across the percolation threshold. Statistical assemblies of conducting elements need only partially fill a surface or volume to obtain conduction. For a surface the percolation threshold is at half-saturation of the sites. For a three-dimensional pore only one-sixth of the sites need be filled. 

Enzymatic studies using selectively addressable fluorescent Rab proteins have elucidated the geranylgeranylation process (16). Structural studies on selectively prenylated Rab proteins have elucidated the exact interaction mechanism of Ras proteins with their transporter protein GDP dissociation inhibitor (GDI) and have led to a membrane extraction model for Rab proteins (58, 59). 

Work has been done to computationally model transporter structure, and this work will gain in value as additional high-resolution three-dimensional membrane protein structures are solved. Similar to the QSAR studies for P-gp described in this chapter, protein structural and substrate affinity modeling approaches have also been applied to various other transporters (181). The resulting three-dimensional structure-function relationships should be useful to understanding how individual genetic differences in transporter function will affect drug transport. 

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