POSTERS - BLAST X - University of Utah

BLAST X Poster #40
LINKING THE TM2 TO HAMP—A TOUGH NUT TO CRACK?
Rachel L. Crowder, Gus A. Wright, and Michael D. Manson
Department of Biology, Texas A&M University, College Station, TX 77843
The HAMP domain is a widely conserved motif found in transmembrane-signaling
proteins in prokaryotes and lower eukaryotes. It consists of a pair of amphipathic helices joined
by a flexible linker. Recently, the solution structure of the Archeoglobus fulgidis Af1503 HAMP
domain was determined using NMR (Hulko et al, Cell 126: 929-940, 2006). The domain forms a
parallel four-helix bundle that packs in a non-canonical knob-on-knob conformation. Several
models have been proposed to explain how the four helix bundle transmits the downward piston
movement of transmembrane 2 (TM2) of E. coli chemoreceptors into the signaling domain to
inhibit CheA kinase activity. The connection between TM2 and the HAMP domain is likely to be
important for transducing the input signal from TM2 to HAMP. We hypothesized that increasing
the flexibility of the connector should attenuate the output signal. To test this idea, residues
between Met-215 Thr-218 of the E. coli aspartate receptor Tar were replaced with four Gly
residues. Gly residues were then deleted (-4G through -1G) and added (+1G through +5G).
Aspartate chemotaxis, rotational bias of tethered cells, mean reversal frequencies of tethered
cells, and in vivo methylation levels were measured. These experiments suggest that increasing
flexibility between TM2 and HAMP strongly biases the receptor to the “off” (CCW-signaling)
state and decreases aspartate mediated signal transmission between TM2 and HAMP.
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