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

ABSTRACT: 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.

SUBMITTER: Ames P

PROVIDER: S-EPMC4154141 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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

Ames Peter P Zhou Qin Q Parkinson John S JS

Molecular microbiology 20131110 5

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 ...[more]

PMID: 24205875

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Project description:UnlabelledThe transmembrane Tsr protein of Escherichia coli mediates chemotactic responses to environmental serine gradients. Serine binds to the periplasmic domain of the homodimeric Tsr molecule, promoting a small inward displacement of one transmembrane helix (TM2). TM2 piston displacements, in turn, modulate the structural stability of the Tsr-HAMP domain on the cytoplasmic side of the membrane to control the autophosphorylation activity of the signaling CheA kinase bound to the membrane-distal cytoplasmic tip of Tsr. A five-residue control cable segment connects TM2 to the AS1 helix of HAMP and transmits stimulus and sensory adaptation signals between them. To explore the possible role of control cable helicity in transmembrane signaling by Tsr, we characterized the signaling properties of mutant receptors with various control cable alterations. An all-alanine control cable shifted Tsr output toward the kinase-on state, whereas an all-glycine control cable prevented Tsr from reaching either a fully on or fully off output state. Restoration of the native isoleucine (I214) in these synthetic control cables largely alleviated their signaling defects. Single amino acid replacements at Tsr-I214 shifted output toward the kinase-off (L, N, H, and R) or kinase-on (A and G) states, whereas other control cable residues tolerated most amino acid replacements with little change in signaling behavior. These findings indicate that changes in control cable helicity might mediate transitions between the kinase-on and kinase-off states during transmembrane signaling by chemoreceptors. Moreover, the Tsr-I214 side chain plays a key role, possibly through interaction with the membrane interfacial environment, in triggering signaling changes in response to TM2 piston displacements.ImportanceThe Tsr protein of E. coli mediates chemotactic responses to environmental serine gradients. Stimulus signals from the Tsr periplasmic sensing domain reach its cytoplasmic kinase control domain through piston displacements of a membrane-spanning helix and an adjoining five-residue control cable segment. We characterized the signaling properties of Tsr variants to elucidate the transmembrane signaling role of the control cable, an element present in many microbial sensory proteins. Both the kinase-on and kinase-off output states of Tsr depended on control cable helicity, but only one residue, I214, was critical for triggering responses to attractant inputs. These findings suggest that signal transmission in Tsr involves modulation of control cable helicity through interaction of the I214 side chain with the cytoplasmic membrane.

Project description:Tsr, the serine chemoreceptor in Escherichia coli, transduces signals from a periplasmic ligand-binding site to its cytoplasmic tip, where it controls the activity of the CheA kinase. To function, Tsr forms trimers of homodimers (TODs), which associate in vivo with the CheA kinase and CheW coupling protein. Together, these proteins assemble into extended hexagonal arrays. Here, we use cryo-electron tomography and molecular dynamics simulation to study Tsr in the context of a near-native array, characterizing its signaling-related conformational changes at both the individual dimer and the trimer level. In particular, we show that individual Tsr dimers within a trimer exhibit asymmetric flexibilities that are a function of the signaling state, highlighting the effect of their different protein interactions at the receptor tips. We further reveal that the dimer compactness of the Tsr trimer changes between signaling states, transitioning at the glycine hinge from a compact conformation in the kinase-OFF state to an expanded conformation in the kinase-ON state. Hence, our results support a crucial role for the glycine hinge: to allow the receptor flexibility necessary to achieve different signaling states while also maintaining structural constraints imposed by the membrane and extended array architecture.IMPORTANCE In Escherichia coli, membrane-bound chemoreceptors, the histidine kinase CheA, and coupling protein CheW form highly ordered chemosensory arrays. In core signaling complexes, chemoreceptor trimers of dimers undergo conformational changes, induced by ligand binding and sensory adaptation, which regulate kinase activation. Here, we characterize by cryo-electron tomography the kinase-ON and kinase-OFF conformations of the E. coli serine receptor in its native array context. We found distinctive structural differences between the members of a receptor trimer, which contact different partners in the signaling unit, and structural differences between the ON and OFF signaling complexes. Our results provide new insights into the signaling mechanism of chemoreceptor arrays and suggest an important functional role for a previously postulated flexible region and glycine hinge in the receptor molecule.

Dataset Information

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

Publications

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

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