New Roles for HAMP Domains: the Tri-HAMP Region of Pseudomonas aeruginosa Aer2 Controls Receptor Signaling and Cellular Localization

Abstract

The Aer2 chemoreceptor from Pseudomonas aeruginosa is an O2 sensor involved in stress responses, virulence, and tuning the behavior of the chemotaxis (Che) system. Aer2 is the sole receptor of the Che2 system. It is soluble, but membrane associated, and forms complexes at the cell pole during stationary phase. The domain arrangement of Aer2 is unusual, with a PAS sensing domain sandwiched between five HAMP domains, followed by a C-terminal kinase-control output domain. The first three HAMP domains form a poly-HAMP chain N-terminal to the PAS sensing domain. HAMP domains are often located between signal input and output domains, where they transduce signals. Given that HAMP1 to 3 reside N-terminal to the input-output pathway, we undertook a systematic examination of their function in Aer2. We found that HAMP1 to 3 influence PAS signaling over a considerable distance, as the majority of HAMP1, 2 and 3 mutations, and deletions of helical phase stutters, led to nonresponsive signal-off or off-biased receptors. PAS signal-on lesions that mimic activated Aer2 also failed to override N-terminal HAMP signal-off replacements. This indicates that HAMP1 to 3 are critical coupling partners for PAS signaling and likely function as a cohesive unit and moveable scaffold to correctly orient and poise PAS dimers for O2-mediated signaling in Aer2. HAMP1 additionally controlled the clustering and polar localization of Aer2 in P. aeruginosa. Localization was not driven by HAMP1 charge, and HAMP1 signal-off mutants still localized. Employing HAMP as a clustering and localization determinant, as well as a facilitator of PAS signaling, are newly recognized roles for HAMP domains.