ASPARAGINE-394 IN PUTATIVE HELIX-11 OF THE GALACTOSE-H-COLI IS ASSOCIATED WITH THE INTERNAL BINDING-SITE FOR CYTOCHALASIN-B AND SUGAR( SYMPORT PROTEIN (GALP) FROM ESCHERICHIA)

The galactose-H+ symport protein (Ga1P) of Escherichia coli is very si milar to the human glucose transport protein, GLUT1, and both contain a highly conserved Asn residue in predicted helix 11 that is different in a cytochalasin B-resistant member of this sugar transport family ( XylE). The role of the Asn(394) residue (which is predicted to be in p utative trans-membrane alpha-helix 11) in the structure/activity relat ionship of the D-galactose-H+ symporter (GalP) was therefore assessed by measuring the interaction of sugar substrates and the inhibitory an tibiotics, cytochalasin B, and forskolin with the wild-type and Asn(39 4) --> Gin mutant proteins, Steady-state fluorescence quenching experi ments show that the mutant protein binds cytochalasin B with a K-d 37- 53-fold higher than the wild type, This low affinity binding was not d etected with equilibrium binding or photolabeling experiments, In cont rast, the mutant protein binds forskolin with a K-d similar to that of the wild type and is photolabeled by 3-I-125-4-azido-phenethylamido - 7-O-succinyl-desacetyl-forskolin. The mutant protein displays an incre ased amount of steady-state fluorescence quenching with the binding of forskolin, suggesting that the substitution of the Asn residue has al tered the environment of a tryptophan, probably Trp(395), in a conform ationally active region of the protein, Time-resolved fluorescence mea surements on the mutant protein provided association and dissociation rate constants (k(2) and k(-2),), describing the initial interaction o f cytochalasin B to the inward-facing binding site (T-i), that are dec reased (9-fold) and increased (4.9-fold) compared with the wild type, This yielded a dissociation constant (K-2) for cytochalasin B to the i nward-facing binding site 44-fold higher than that of the wild type, T he binding of forskolin gave values for k(2) and k(-2) 3.9- and 3.6-fo ld lower, respectively, yielding a K-2 value for T-i similar to that o f the wild type, The low overall affinity (high K-d) of the mutant pro tein for cytochalasin B is due mainly to a disruption in binding to th e T-i conformation. It is proposed that Asn(394) forms either a direct binding interaction with cytochalasin B or is part of the immediate e nvironment of the binding site and that Asn(394) is in the immediate e nvironment, but not part, of the forskolin binding site, The ability o f the mutant protein to catalyze energized transport is only mildly im paired with 4.8- and 2.1-fold reduction in V-max/K-m values for D-gala ctose and D-glucose, respectively, In stark contrast, the overall K-d describing binding of D-galactose and D-glucose to the inward-facing c onformation of the mutant and their subsequent trans location across t he membrane is substantially increased (64-fold for D-galactose and 16 3.3-fold for D-glucose), These data indicate that Asn(394) is associat ed with both the cytochalasin B and internal sugar binding sites, This conclusion is also supported by data showing that the sugar specifici ty of the mutant protein has been altered for D-xylose, This work powe rfully illustrates how comparisons of the aligned amino acid sequences of homologous membrane proteins of unknown structure and characteriza tion of their phenotypes can be used to map substrate and ligand bindi ng sites.