POSTERS - BLAST X - University of Utah

BLAST X Wed. Morning Session
THE TM2-HAMP CONNECTION
Gus A. Wright, Rachel L. Crowder and Michael D. Manson
Department of Biology, Texas A&M University, College Station, TX 77843
The HAMP domain—a conserved protein motif in most Histidine kinases, Adenylate
cyclases, Methyl-accepting chemotaxis proteins, and Phosphatases—typically conforms to a
helix–connector-helix domain architecture. Recently, an NMR solution structure was determined
for the Af1503 HAMP from the archaebacteria Archeoglobus fulgidis (Hulko, et al, Cell 126: 929-
940, 2006). This structure revealed that the HAMP domain is a parallel four-helix bundle with a
knob-on-knob packing. Similar structures of HAMP domains are proposed to exist in all 5
chemoreceptors of Escherichia coli, including the aspartate chemoreceptor Tar. The HAMP
domain of Tar receives information from TM2 and regulates the transmission of the signal to the
kinase signaling domain. Mutations were introduced into the TM2-HAMP connector region of
Tar to determine if manipulating the input signal into the HAMP affects the output signal to the
signaling domain. MLLT residues between R214 and P219 were deleted (-4 through -1), and
additional LLT tandem repeats were added up to 8 residues after P219 (+1 through +8).
Aspartate sensitivity, rotational bias, mean reversal frequency, and in vivo methylation were
measured for these mutants. The results suggest that adding helical turns in this region
destabilizes, or “relaxes”, the HAMP/signaling domains; while, removing helical turns from this
region stabilizes, or “tightens”, the HAMP/signaling domains. In order to determine if increasing
flexibility of the TM2/HAMP connector affects the output signal, a second set of mutants were
constructed to replace the MLLT region with four tandem glycine residues (4G). Glycine
residues were then subtracted (-4G through -1G) and added (+1G through +5G). Data collected
from this experiment suggests that increasing the flexibility of this region dampens the input
signal from TM2, in addition to destabilizing the HAMP/signaling domains.
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