The kinetics and thermodynamics of the binding of cytochalasin B to sugar transporters.
The kinetics of the binding of cytochalasin B to the proton-linked L-arabinose (AraE) and D-galactose (GalP) symporters from Escherichia coli and to the human erythrocyte glucose transporter (GLUT1) have been investigated by exploiting the changes in protein fluorescence that occur upon binding the ligand. Steady-state measurements yielded Kd values of 1.1, 1.9 and 0.14 microM for the AraE, GalP and GLUT1 proteins, respectively. The association and dissociation rate constants for the binding of cytochalasin B have been determined by stopped-flow spectroscopy. In each case, the apparent Kd was calculated from the corresponding rate constants, yielding values of 1.5, 0.4 and 1.6 microM for AraE, GalP and GLUT1, respectively. The differences between these apparent Kd values and those measured by fluorescence titration is interpreted in terms of the following three step mechanism where CB represents cytochalasin B: [formula: see text] The transporter is proposed to alternate between two different conformational forms (T1 and T2), with cytochalasin B binding only to the T2 conformation, to induce a further conformational transition of the transporter to the T3 form. The values for the overall dissociation constants show that the T1 conformation is favoured by AraE and GalP in the absence of ligands, but the T2 conformation is favoured by GLUT1. Thus, the binding of cytochalasin B to GLUT1 alters the equilibrium towards the T3(CB) conformational state, producing the observed tight binding, in contrast to the changes in the equilibrium observed with the binding of cytochalasin B to AraE and GalP. A thermodynamic analysis of these conformational transitions has been performed. The T1 and T2 conformations may represent transporter states in which the binding site is facing outwards and inwards, respectively.
Walmsley
A R
AR
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, England.
Lowe
A G
AG
Henderson
P J
PJ
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D003571:Cytochalasin B / Q000737:chemistry / Q000378:metabolism*; D004912:Erythrocytes / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D051272:Glucose Transporter Type 1; D006801:Humans; D007700:Kinetics; D008968:Molecular Conformation; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D033902:Periplasmic Binding Proteins; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
C016242:AraF protein, E coli; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D051272:Glucose Transporter Type 1; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; C495677:SLC2A1 protein, human; C023550:galactose-binding protein; D003571:Cytochalasin B