Abstract
HAMP domains are dimeric, four-helix bundles that transduce conformational signals in bacterial receptors. Genetic studies of the Escherichia coli serine receptor (Tsr) provide an opportunity to understand HAMP conformational behavior in terms of functional output. To increase its stability, the Tsr HAMP domain was spliced into a poly-HAMP unit from the Pseudomonas aeruginosa Aer2 receptor. Within the chimera, the Tsr HAMP undergoes a thermal melting transition at a temperature much lower than that of the Aer2 HAMP domains. Pulse-dipolar electron spin resonance spectroscopy and site-specific spin-labeling confirm that the Tsr HAMP maintains a four-helix bundle. Pulse-dipolar electron spin resonance spectroscopy was also used to study three well-characterized HAMP mutational phenotypes: those that cause flagella rotation that is counterclockwise (CCW) A and kinase-off; CCW B and also kinase-off; and, clockwise (CW) and kinase-on. Conformational properties of the three HAMP variants support a biphasic model of dynamic bundle stability, but also indicate distinct conformational changes within the helix bundle. Functional kinase-on (CW) and kinase-off (CCW A) states differ by concerted changes in the positions of spin-label sites at the base of the bundle. Opposite shifts in the subunit separation distances of neighboring residues at the C-termini of the α1 and α2 helices are consistent with a helix scissors motion or a gearbox rotational model of HAMP activation. In the drastic kinase-off lesion of CCW B, the α1 helices unfold and the α2 helices form a tight two-helix coiled-coil. The substitution of a critical residue in the Tsr N-terminal linker or control cable reduces conformational heterogeneity at the N-terminus of α1 but does not affect structure at the C-terminus of α2. Overall, the data suggest that transitions from on- to off-states involve decreased motional amplitudes of the Tsr HAMP coupled with helix rotations and movements toward a two-helix packing mode.