The effect of individual positively charged residues on the orientation in the membrane was analyzed in three dual-topology transporters of the small. multidrug resistance (SMR) family: AAVE4701aave of Acidovorax avenae, EMREecol of Escherichia coli, and RRUA0272rrub of Rhodospirillum rubrum. It is shown that (i) individual positive charges have different impacts on the orientation, (ii) positive charges that are conserved in the three different proteins do not have the same impact on the orientation, (iii) positive charges in odd- and even-numbered loops have different impacts, (iv) for some, but not all, the impact depends on the presence of other positive charges, and (v) proteins from which all positive charges are removed in some cases are dual-topology proteins and in other cases have a single orientation. A small number of positive charges placed in the loops of the latter proteins results in the violation of the so-called positive-inside rule that has been reported previously [Kolbusz, M. A., et al. (2010) J. Mol. Biol. 402, 127-138]. We conclude that each positive charge shifts the distribution between the two orientations toward the state that has the positive charge in the cytoplasm but that intrinsic factors other than positive charges determine the orientation as well. The ability of the mutants of AAVE4701aave and EMREecol to confer resistance against ethidium bromide revealed an essential role in catalysis for a conserved pair of positive charges in the second loop. No significant relation between activity and the relative orientation of the monomeric subunits in the dimer could be demonstrated.
- PROTEIN EVOLUTION
- OLIGOMERIC STATE