Twenty cytochalasins were tested for binding to and for inhibition of glucose transport in human erythrocyte membrane. In this membrane three cytochalasin B (CB) binding sites have been identified. All but three of the cytochalasins bind at site II. On the other hand, only nine of them, which are structurally closely related, bind at site I and inhibit glucose transport. For site I (and site III) binding and glucose transport inhibitory activities (a) the macrocyclic ring in the cytochalasin molecule must be at least 13-membered, (b) the nature of the aromatic ring at C-10 is not important, (c) the C-20-C-23 region makes a major contribution, and (d) the C-5-C-7 segment has a relatively minor influence. These findings do not support a proposed mechanism which involves 24, C-23, C-20, and C-1 oxygen atoms for interaction of CB with glucose carrier. The structural requirements for site II activity are less stringent. The size and the structure of the macrocyclic ring and the nature of the aromatic residue at C-10 modulate this activity only slightly, if at all. Modifications in the C-5-C-7 region of the molecule, however, result in substantial changes in this activity.

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On the basis of details of the three-dimensional structures of fi-D-glucose and of cytochalasins, either previously published or reported here (cytochalasin A), we propose a model to explain the observed difference in activity of cytochalasins in the inhibition of glucose transport. In our model cytochalasin B binds to the glucose carrier through hydrogen bonds at N2 (donates), 07 (accepts), and 023 (accepts) analogous to 06, 03, and 01, respectively, on 8-D-glucose. The hydrophobic region from C13 to C19 is also essential in binding and appears to act as an anchor in a hydrophobic domain of the glucose carrier. The presence of hydrophilic groups in this essential hydrophobic region accounts, at least in part, for the inactivity of the other cytochalasins in the series.Cytochalasin B (CB) is a remarkably specific and potent inhibitor of D-glucose transport in human erythrocytes. The inhibition is kinetically of the competitive type (1) and may or may not be isosteric at the molecular level. The inhibitor binds to the erythrocyte membrane at three different receptor sites. The binding to one of these (site I) is specifically displaced by Dglucose. Ample evidence now available indicates that this glucose-sensitive site I binding is responsible for the inhibition, and this receptor protein is considered to be the glucose transport carrier itself (1)(2)(3)(4). In all the cytochalasins tested, the site I binding activity accompanies glucose transport inhibitory activity (Table 1) (4). A study (4) on the structure-activity relationship of a series of cytochalasins both as transport inhibitors and as ligands for the site I binding has shown that the specificity is indeed very narrow: for both the activities, the size and the structure of the macrocycle in the C20 to C23 region and the substitution pattern in the C5 to C7 segment are important. However, to understand the mechanism of interaction or lack of interaction of cytochalasins with the glucose carrier, the role of each functional group in these segments should be delineated.In addition to investigating structural requirements of a receptor site through binding studies, one can examine three-dimensional structural specificity by studying the crystallographically determined molecular structures and correlating structural features with the activity data. Seven cytochalasin structures have been determined previously (Table 1) of which only one, CB, exhibits these activities. We have determined the crystal structure of cytochalasin A (CA), which is also site I active, in order to distinguish the stereo features responsible for conferring glucose transport inhibitory and site I binding activities-i. e., those common to CA and CB and not found in the inactive cytochalasins. (8) A, a = y = 90°, 3 = 114.457(4)°, volume = 1,378.1 K3, Z = 2. Three-dimensional x-ray intensity data were collected on an Enraf-Nonius (Bohemia, NY) CAD-4 automated diffractometer using copper radiation by the 0 -20 scan technique; 3,050 independent data were collected, of which 2,763 w...

A family of structurally related intrinsic membrane proteins (facilitative glucose transporters) catalyzes the movement of glucose across the plasma membrane of animal cells. Evidence indicates that these proteins show a common structural motif where approximately 50% of the mass is embedded in lipid bilayer (transmembrane domain) in 12 alpha-helices (transmembrane helices; TMHs) and accommodates a water-filled channel for substrate passage (glucose channel) whose tertiary structure is currently unknown. Using recent advances in protein structure prediction algorithms we proposed here two three-dimensional structural models for the transmembrane glucose channel of GLUT1 glucose transporter. Our models emphasize the physical dimension and water accessibility of the channel, loop lengths between TMHs, the macrodipole orientation in four-helix bundle motif, and helix packing energy. Our models predict that five TMHs, either TMHs 3, 4, 7, 8, 11 (Model 1) or TMHs 2, 5, 11, 8, 7 (Model 2), line the channel, and the remaining TMHs surround these channel-lining TMHs. We discuss how our models are compatible with the experimental data obtained with this protein, and how they can be used in designing new biochemical and molecular biological experiments in elucidation of the structural basis of this important protein function.