THE CONSERVED MOTIF, GXXX(D E)(R/K)XG[X](R/K)(R/K), IN HYDROPHILIC LOOP-2/3 OF THE LACTOSE PERMEASE/

A conserved motif, GXXX(D/E)(R/K)XG(R/K)(R/K), has been identified amo ng a large group of evolutionarily related membrane proteins involved in the transport of small molecules across the membrane. To determine the importance of this motif within the lactose permease of Escherichi a coli, a total of 28 site-directed mutations at the conserved first, fifth, sixth, eighth, ninth, and tenth positions were analyzed. A dram atic inhibition of activity was observed with all bulky mutations at t he first-position glycine. Based on these results, together with seque nce comparisons within the superfamily, it seems likely that small sid e chain volume (and possibly high beta-turn propensity) may be structu rally important at this position. The acidic residue at the fifth posi tion was also found to be very important for transport activity and ev en a conservative glutamate at this location exhibited marginal transp ort activity, In contrast, many substitutions at the eighth-position g lycine, even those with a high side chain volume and/or low beta-turn propensity, still retained high levels of transport activity. Similarl y, none of the basic residues within the motif were essential for tran sport activity when replaced individually by nonbasic residues. Howeve r, certain substitutions at the basic residue sites as well as the eig hth-position glycine were observed to have moderately reduced levels o f active transport of lactose, Taken together, the results of this stu dy confirm the importance of the conserved loop 2/3 motif in transport function. It is suggested that this moth may be important in promotin g global conformational changes within the permease.