Mutational Replacements at the “Glycine Hinge” of the <i>Escherichia coli</i> Chemoreceptor Tsr Support a Signaling Role for the C-Helix Residue

Pedetta, Andrea; Massazza, Diego A.; Seitz, María Karina Herrera; Studdert, Claudia A.

doi:10.1021/acs.biochem.7b00455

Cited by 11 publications

(13 citation statements)

References 36 publications

(78 reference statements)

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“…The functional role of the glycine hinge in chemoreceptor signaling has been a topic of much speculation and is somewhat controversial. Mutational analyses have shown that side chain replacements at the glycine hinge residues impair or abrogate chemotaxis ability (22, 47). Moreover, several studies have suggested that the glycine hinge introduce structural flexibility to the helix bundle, perhaps to allow bending (14, 22).…”

Section: Discussionmentioning

confidence: 99%

In Situ Conformational Changes of the Escherichia coli Serine Chemoreceptor in Different Signaling States

Yang

Cassidy

Ames

et al. 2019

mBio

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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.

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Section: Discussionmentioning

confidence: 99%

In Situ Conformational Changes of the Escherichia coli Serine Chemoreceptor in Different Signaling States

Yang

Cassidy

Ames

et al. 2019

mBio

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“…9A). MH bundle conformational changes, in turn, probably elicit-through an intervening flexible region and glycine hinge-opposing stability shifts in the receptor hairpin tip that modulate CheA kinase activity (24,31,55,56).…”

Section: Discussionmentioning

confidence: 99%

“…(B) Primary structure of the Tsr MH bundle cap. The sequence logo depicts the predominant residues at each MH cap position in 2,428 nonredundant members of the 36H class of chemoreceptors (31,56,81). The corresponding Tsr residues are listed between the MH1 and MH2 logos.…”

Section: Significancementioning

confidence: 99%

A zipped-helix cap potentiates HAMP domain control of chemoreceptor signaling

Flack

Parkinson

2018

Proc. Natl. Acad. Sci. U.S.A.

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Environmental awareness is an essential attribute for all organisms. The chemotaxis system of provides a powerful experimental model for the investigation of stimulus detection and signaling mechanisms at the molecular level. These bacteria sense chemical gradients with transmembrane proteins [methyl-accepting chemotaxis proteins (MCPs)] that have an extracellular ligand-binding domain and intracellular histidine kinases, adenylate cyclases, methyl-accepting proteins, and phosphatases (HAMP) and signaling domains that govern locomotor behavior. HAMP domains are versatile input-output elements that operate in a variety of bacterial signaling proteins, including the sensor kinases of two-component regulatory systems. The MCP HAMP domain receives stimulus information and in turn modulates output signaling activity. This study describes mutants of the serine chemoreceptor, Tsr, that identify a heptad-repeat structural motif (LLF) at the membrane-proximal end of the receptor signaling domain that is critical for HAMP output control. The homodimeric Tsr signaling domain is an extended, antiparallel, four-helix bundle that controls the activity of an associated kinase. The N terminus of each subunit adjoins the HAMP domain; the LLF residues lie at the C terminus of the methylation-helix bundle. We found, by using in vivo Förster resonance energy transfer kinase assays, that most amino acid replacements at any of the LLF residues abrogate chemotactic responses to serine and lock Tsr output in a kinase-active state, impervious to HAMP-mediated down-regulation. We present evidence that the LLF residues may function like a leucine zipper to promote stable association of the C-terminal signaling helices, thereby creating a metastable helix-packing platform for the N-terminal signaling helices that facilitates conformational control by the HAMP domains in MCP-family chemoreceptors.

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“…Mutational analyses of the amino acyl residues at the two hinges have shown that chemotaxis ability is reduced by mutational substitutions at these loci. Substitution of cysteine or alanine for the glycines in the glycine hinge greatly reduced chemotaxis, as well as kinase activation or kinase control through ligand occupancy (28), and a recent, more extensive, mutational analysis observed similar effects of other substitutions (34). The two-helix region of the HAMP hinge corresponds to residues T262, D263, and T264 in Tar (31).…”

Section: Indications That Chemoreceptors Have Flexible Hingesmentioning

confidence: 94%

“…An evolutionary genomics study of Ͼ2,000 sequences of chemoreceptor cytoplasmic domains from Ͼ150 bacterial and archaeal species, reflecting bacterial and archaeal taxonomic diversity, identified the region surrounding the glycine hinge as a conserved flexible bundle subdomain and thus a feature likely to have an important role in chemoreceptor function and/or structure (29). Recent analysis of Ͼ22,500 chemoreceptor sequences from 5,820 species confirmed this identification (34). The observations documented in our current study demonstrate that intact functional chemoreceptors indeed bend at the glycine hinge.…”

Section: Indications That Chemoreceptors Have Flexible Hingesmentioning

confidence: 97%

Flexible Hinges in Bacterial Chemoreceptors

et al. 2018

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Transmembrane bacterial chemoreceptors are extended, rod-shaped homodimers with ligand-binding sites at one end and interaction sites for signaling complex formation and histidine kinase control at the other. There are atomic-resolution structures of chemoreceptor fragments but not of intact, membrane-inserted receptors. Electron tomography of signaling complex arrays lack distinct densities for chemoreceptor rods away from the well-ordered base plate region, implying structural heterogeneity. We used negative staining, transmission electron microscopy, and image analysis to characterize the molecular shapes of intact homodimers of the aspartate receptor Tar rendered functional by insertion into nanodisc-provided lipid bilayers. Single-particle analysis plus tomography of particles in a three-dimensional matrix revealed two bend loci in the chemoreceptor cytoplasmic domain, (i) a short, two-strand gap between the membrane-proximal, four-helix-bundle HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemoreceptors, and phosphatases) domain and the membrane-distal, four-helix coiled coil and (ii) aligned glycines in the extended, four-helix coiled coil, the position of a bend noted in the previous X-ray structure of a receptor fragment. Our images showed HAMP bends from 0° to ∼13° and glycine bends from 0° to ∼20°, suggesting that the loci are flexible hinges. Variable hinge bending explains indistinct densities for receptor rods outside the base plate region in subvolume averages of chemotaxis arrays. Bending at flexible hinges was not correlated with the chemoreceptor signaling state. However, our analyses showed that chemoreceptor bending avoided what would otherwise be steric clashes between neighboring receptors that would block the formation of core signaling complexes and chemoreceptor arrays. This work provides new information about the shape of transmembrane bacterial chemoreceptors, crucial components in the molecular machinery of bacterial chemotaxis. We found that intact, lipid-bilayer-inserted, and thus functional homodimers of the chemoreceptor Tar exhibited bends at two flexible hinges along their ∼200-Å, rod-like, cytoplasmic domains. One hinge was at the short, two-strand gap between the membrane-proximal, four-helix-bundle HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemoreceptors, and phosphatases) domain and the membrane-distal, four-helix coiled coil. The other hinge was at aligned glycines in the extended, four-helix coiled coil, where a bend had been identified in the X-ray structure of a chemoreceptor fragment. Our analyses showed that flexible hinge bending avoided structural clashes in chemotaxis core complexes and their arrays.

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Mutational Replacements at the “Glycine Hinge” of the Escherichia coli Chemoreceptor Tsr Support a Signaling Role for the C-Helix Residue

Cited by 11 publications

References 36 publications

In Situ Conformational Changes of the Escherichia coli Serine Chemoreceptor in Different Signaling States

In Situ Conformational Changes of the Escherichia coli Serine Chemoreceptor in Different Signaling States

A zipped-helix cap potentiates HAMP domain control of chemoreceptor signaling

Flexible Hinges in Bacterial Chemoreceptors

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