TRANSMEMBRANE ALPHA-HELIX INTERACTIONS ARE REQUIRED FOR THE FUNCTIONAL ASSEMBLY OF THE ESCHERICHIA-COLI TOL COMPLEX

TolQ, TolR and TolA are membrane proteins involved in maintaining the structure of Escherichia coli cell envelope. TolQ and TolR span the in ner membrane with three and with one a-helical segments, respectively. The tolQ925 mutation (A177V),located in the third putative transmembr ane helix of TolQ (TolQ-III), induces cell sensitivity to bile salts a nd tolerance towards colicin A but not colicin El, unlike a null tolQ mutation, which induces tolerance to all group A colicins. Since TolQ is required for colicin A and Fl uptake, in contrast to TolR, which is necessary only for colicin A, we hypothesized that the tolQ925 mutati on might affect an interaction between TolQ and TolR. We therefore sea rched for suppressor mutations in TolR that would restore cell envelop e integrity and colicin A sensitivity to the tolQ925 mutant. Five diff erent tolR alleles were isolated and characterized. Four of these supp ressor mutations were found to be clustered in the single putative tra nsmembrane helix of TolR (TolR-I) and one was located at the extreme C terminus of the protein. In addition, we isolated a spontaneous intra genic suppressor localized in the first transmembrane helix of TolQ (T olQ-I). These observations strongly suggest that TolR and TolQ interac t via their transmembrane segments. Sequence analysis indicates that A la177 lies on the ex-helix face of TolQ-III that, according to its com position and evolutionary conservation, is the most likely to be invol ved in protein/protein interaction. Energy minimization of atomic mode ls of the wild-type and mutated forms of TolQ-III and TolR-I suggests that the deleterious effect of the A177V substitution arises from a di rect steric hindrance of this residue with neighboring transmembrane s egments, and that suppressor mutations may alleviate this effect eithe r directly or indirectly, e.g. by affecting the stability of conformat ional equilibrium of the transmembrane region of the complex.