Signal Transduction Mechanisms of HAMP and PAS Domains in Bacterial Chemotaxis

Abstract

Bacteria utilize two-component systems to respond and adapt to changes in their environments. Central to the systems are modular receptors that comprise various functional domains to detect those changes and relay signals to effector domains. HAMP (Histidine kinases, Adenylate cyclases, Methyl accepting proteins and Phosphatases) and PAS (Per–Arnt–Sim) are two of the most common domains that couple various effectors to regulate a wide range of cellular activities. HAMP domains are signal relay modules that connects input and output domains. The HAMP domain from the E. coli serine receptor Tsr has been extensively studied by using genetic techniques, which leads to a model of HAMP biphasic stability that explains the behaviors of Tsr mutant receptors. However, limited biophysical data on the Tsr HAMP are available due to the instability of the domain. In order to provide stability to the Tsr HAMP, a chimera containing Tsr spliced into the poly-HAMP domains from Pseudomonas aeruginosa Aer2 (PaAer2) was created. Within the chimera, the Tsr HAMP maintains its characteristic four-helix coiled-coil structure with the distinctively lowered melting temperature compared to the PaAer2. This chimera was used to study three well-characterized HAMP mutational phenotypes differentiated by flagella-rotation patterns and CheA kinase activities: functional counterclockwise flagella rotation [CCW(A), kinase off], functional clockwise flagella rotation (CW, kinase on), and lesion-induced counterclockwise rotation [CCW(B), kinase off]. The stabilities and structural dynamics of the three phenotypes conform to the biphasic model. The transitions between functional on and off states are mediated by helix rotations and scissor-type movements. In the lesion-induced kinase off, the AS1 helices dissociate from the bundle while the AS2 helices form a two-helix colied coil. Overall, this study provides insights into relationships between HAMP conformational behaviors and their corresponding functional outputs. PAS domains are sensor motifs that are critical in signal transductions of prokaryotic and eukaryotic sensory proteins including chemoreceptors. Vibrio cholerae Aer2 (VcAer2), a PaAer2 homolog, has been shown to mediate responses to oxygen through the heme-binding PAS domains. Substitution of the conserved Trp 276 in the PAS2 domain to Leu abolished the O2-stabilizing ability, which corroborates its O2-ligating role. The crystal structure of the VcAer2 W276L is highly similar to the CN-bound PAS domain from PaAer2, suggesting the structure of the W276L mutant might represent the ligand-binding state. VcAer2 can serve as a promising alternative to E. coli Aer or PaAer2 for investigating PAS-mediated chemotaxis