HAMP domain structural determinants for signalling and sensory adaptation in Tsr, the Escherichia coli serine chemoreceptor.

HAMP domains mediate input-output transactions in many bacterial signalling proteins. To clarify the mechanistic logic of HAMP signalling, we constructed Tsr-HAMP deletion derivatives and characterized their steady-state signal outputs and sensory adaptation properties with flagellar rotation and receptor methylation assays. Tsr molecules lacking the entire HAMP domain or just the HAMP-AS2 helix generated clockwise output signals, confirming that kinase activation is the default output state of the chemoreceptor signalling domain and that attractant stimuli shift HAMP to an overriding kinase-off signalling state to elicit counter-clockwise flagellar responses. Receptors with deletions of the AS1 helices, which free the AS2 helices from bundle-packing constraints, exhibited kinase-off signalling behaviour that depended on three C-terminal hydrophobic residues of AS2. We conclude that AS2/AS2' packing interactions alone can play an important role in controlling output kinase activity. Neither kinase-on nor kinase-off HAMP deletion outputs responded to sensory adaptation control, implying that out-of-range conformations or bundle-packing stabilities of their methylation helices prevent substrate recognition by the adaptation enzymes. These observations support the previously proposed biphasic, dynamic-bundle mechanism of HAMP signalling and additionally show that the structural interplay of helix-packing interactions between HAMP and the adjoining methylation helices is critical for sensory adaptation control of receptor output.