Non-native interactions within the coiled-coil cytoplasmic domain of chemoreceptors are compatible with signaling

KARINA HERRERA SEITZ; ANDREA PEDETTA; CLAUDIA STUDDERT

Rosario

Congreso; IX Congreso de Microbiología General; 2013

Sociedad Argentina de Microbiología General (SAMIGE)

Bacterial chemoreceptors usually detect extracellular signals through a periplasmic sensing domain and transmit them to a highly conserved intracellular domain. The signal then reaches the flagellar motors to control swimming behavior. The cytoplasmic signaling domain consists of a long alpha-helical hairpin that forms, in the dimer, a coiled-coil four-helix bundle. The huge variety of chemoreceptors identified from genomic analyses in Bacteria and Archaea can be classified into a small number of classes according to the length of their cytoplasmic domain. Differences in length are due to the presence of pairs of insertions or deletions (indels) of seven-residue stretches or heptads, located symmetrically with respect to the hairpin turn. The five E. coli chemoreceptors belong to the 36H-class since they possess 36 heptads in this domain. The size and location of the indels highlight the importance of the coiled-coil structure and suggest the existence of specific interactions between the two arms of the hairpin, that are needed to preserve proper signal transmission. To understand the structural requirements of signal transmission that led to this peculiar evolution pattern, in our lab we engaged in the construction and characterization of derivatives of the serine chemoreceptor of E. coli, Tsr, with altered heptad content. In previous work, we had obtained a functional derivative of Tsr whose cytoplasmic hairpin had been shortened by a symmetric pair of seven-residue deletions. This construct mimics natural chemoreceptors of the 34H-class, since the deletions are located at a certain position (level I) and they do not alter the native interactions between the two antiparallel helices along the rest of the cytoplasmic hairpin. In this work, we assessed the effect of altering native interactions within the coiled-coil. To that end, we made a new pair of symmetric seven-residue deletions at a different position of the hairpin (level II). Again, we were able to find functional derivatives of this new 34H-class derivative. Afterwards, we designed constructs with crossed combinations of the deletions (level I on the N-arm, level II on the C-arm, and the opposite), so that the remaining heptad of each level would interact with each other, in a non-native fashion. Even though the constructs showed no Tsr function, in both cases we were able to obtain functional derivatives upon random mutagenesis that generated single point mutations. The ability of the constructs to tolerate this alteration in the partner interaction between heptads indicates that there is no strict specificity along the whole C- and N-helices of the antiparallel hairpin. The observed symmetry in the indels along evolution might rather reflect the need to preserve certain crucial N- to C-interactions in order to attain proper signaling behavior of the chemoreceptor. We are currently trying to identify such determinants.