TSR CONSTRUCTIONS WITH SYMMETRIC HEPTAD DELETIONS DISPLAY FULL FUNCTION

MASSAZZA, DA; IZZO, SA; STUDDERT, CA

New Orleans

Congreso; XIth BLAST (Bacterial Locomotion and Signal Transduction) Meeting; 2011

BLAST

Methyl-accepting chemotaxis proteins (MCPs) share a highly conserved cytoplasmic domain. It consists in a long alpha-helical hairpin that forms, in the dimer, a coiled coil four-helix bundle. A comparison between MCPs from different microorganisms has identified the existence of insertions/deletions of seven aminoacids (heptads) in a symmetric arrangement, that is, situated at similar distances from the hairpin tip. This observation suggests that specific interactions between the helices in the coiled coil arrangement are essential for function. The whole number of heptads (ranging from 28 to 44) in this specific domain, plus certain sequence conservation, have been used to define seven classes of MCPs. Interestingly, some microorganisms possess MCPs belonging to different classes, raising the question of whether they cluster together and form mixed arrangements, or if they need to be segregated in the cell for proper function. We reasoned that making a shortened Tsr derivative, intended to mimic an MCP from a shorter class, could provide an experimental system for exploring these issues. Accordingly, we introduced heptad deletions into one, the other or both helices of Tsr, the E.coli chemoreceptor for serine (36-heptad class). The location of the deletions was chosen based in an alignment with a chemoreceptor from R. sphaeroides, that naturally lacks a heptad in each helix and thus belongs to the 34-heptad class of MCPs. We analyzed the effect of the deletions on several functional/structural aspects of Tsr: - chemotaxis to serine in soft agar plates - kinase activation (pseudotaxis and tethering assays) - response to serine by changing the direction of flagellar rotation in a CW-biased strain - adaptation to serine in free-swimming assays - methylation response after attractant stimulus in the absence of CheA and CheW - trimer formation ability by in vivo crosslinking assays While single asymmetric deletions abolished serine chemotaxis and caused serious alterations in several wild type Tsr abilities, the double symmetric deletion generated a receptor with partial function and apparently normal higher order interactions. Moreover, point mutations in this doubly deleted construction, but not in the singly deleted ones, rendered fully functional Tsr derivatives. Interestingly, one of such mutations replaces an isoleucine, conserved in the 36H class to which Tsr belongs, for a valine, a residue that is highly conserved at that position in the 34H class, to which the shortened version mimics. These results highlight the importance of the coiled coil arrangement for a proper transmission of the signal from the periplasmic domain of Tsr to the tip of the cytoplasmic domain. The location and effect of mutations that restore function to the shortened version of the receptor might help to identify key features required for this transmission.