Function and coiled-coil interactions in the cytoplasmic domain of Tsr chemoreceptor and their variants.

FERNANDO E. HERRERA; A. SERGIO GARAY; CLAUDIA A. STUDDERT; DANIEL E. RODRIGUES

San Miguel de Tucumán

Congreso; IIILAFeBS, IX IberoAmerican congress of Biophysics, XLV SAB Annual Meeting; 2016

Sociedad Argentina de Biofísica

When swimming in a chemoattractant gradient, bacteria perform a temporal comparison of the molecular concentration via its membrane-bound chemoreceptors (e.g. Tar, Tsr) that act on the Che pathway modulating the switching frequency of the flagellar motors, which in turn sets the tumbling frequency. Therefore, runs are in average longer in the favorable direction and the resulting biased random walk drives the bacterium up the chemoattractant gradient. Beyond the periplasmic ligand-binding domain, the cytoplasmic region consist of a HAMP domain followed by an α-helical hairpin that forms in the dimer a coiled-coil four helix bundle. The signal transmission through this structure is an open question. Experiments to analyze the activity of different variants (?constructs?) of the cytoplasmic domain of this chemoreceptor (e.g. mutations, heptad deletions, insertions) had been performed ? to clarify the mechanism of signal transmission. We have obtained one hundred 3D structures of each of the WT dimerichelical hairpin Tsr and several constructs by comparative modeling. We analyzed over each of these sets the physicochemical effects of the modifications of the wild sequence using the knob-into-hole paradigm of coiled-coil structures, the comparison among the root mean square fluctuations of the structures and the contributions to the desolvation and electrostatic free energy terms of pairs of residues in each construct. The sampling over a set of 100 structures for each construct allows for the calculation of the likelihood of existence of knobs making the analysis more robust. We found that some constructs that loses their kinase activation ability lack the Q298, L302, V316 and N319 conserved knobs. Concomitantly the likelihood of knobs in the protein interaction region is enhanced. The V347M mutation that restores the kinase activation ability on one construct, shows a lower probability of knobs inthe protein interaction zone.