Deducing the organization of a transmembrane domain by disulfide cross-linking. The bacterial chemoreceptor Trg.

Lee, G F; Burrows, Gregory G.; Lebert, Michael; Dutton, David P.; Hazelbauer, Gerald L.

doi:10.1016/s0021-9258(18)43969-5

Cited by 101 publications

(34 citation statements)

References 29 publications

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“…The mechanism of transmembrane signaling utilized by the aspartate receptor is likely to be a general feature of structurally and functionally similar receptors which regulate histidine kinase activity. Evidence for such generality is provided by the construction of an active chimeric receptor linking the aspartate receptor's ligand binding and transmembrane domains to the cytoplasmic histidine kinase domain of the EnvZ receptor, a related bacterial protein responsible for osmosensing (Utsumi et al, 1989) bacterial ribose and galactose receptor exhibiting high homology to the aspartate receptor, have yielded results consistent with the signaling role of the second membranespanning helix proposed in Figure 5 (Lee et al, 1995(Lee et al, ,1994.…”

Section: Discussionmentioning

confidence: 68%

Transmembrane signaling by the aspartate receptor: Engineered disulfides reveal static regions of the subunit interface

Chervitz¹,

Lin²,

Falke³

1995

Biochemistry

199

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Ligand binding to the periplasmic domain of the transmembrane aspartate receptor generates an intramolecular conformational change which spans the bilayer and ultimately signals the cytoplasmic CheA histidine kinase, thereby triggering chemotaxis. The receptor is a homodimer stabilized by the interface between its two identical subunits: the present study investigates the role of the periplasmic and transmembrane regions of this interface in the mechanism of transmembrane signaling. Free cysteines and disulfide bonds are engineered into selected interfacial positions, and the resulting effects on the transmembrane signal are assayed by monitoring in vitro regulation of kinase activity. Three of the 14 engineered cysteine pairs examined, as well as six of the 14 engineered disulfides, cause perturbations of the interface structure which essentially destroy transmembrane regulation of the kinase. The remaining 11 cysteine pairs, and eight engineered disulfides covalently linking the two subunits at locations spanning positions 18-75, are observed to retain significant transmembrane kinase regulation. The eight functional disulfides positively identify adjacent faces of the two N-terminal helices in the native receptor dimer and indicate that large regions of the periplasmic and transmembrane subunit interface remain effectively static during the transmembrane signal. The results are consistent with a model in which the subunit interface plays a structural role, while the second membrane-spanning helix transmits the ligand-induced signal across the bilayer to the kinase binding domain. The effects of engineered cysteines and disulfides on receptor methylation in vitro are also measured, enabling direct comparison of the in vitro methylation and phosphorylation assays.

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Section: Discussionmentioning

confidence: 68%

Transmembrane signaling by the aspartate receptor: Engineered disulfides reveal static regions of the subunit interface

Chervitz¹,

Lin²,

Falke³

1995

Biochemistry

199

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“…The α carbons of disulfide-bonded cysteines in proteins are less than 7 Å from each other ( , ). The rate of disulfide bond formation has been used to indicate the relative distance between the reactive cysteines in previous structural studies using cysteine scanning and oxidative cross-linking methods ( , ). Although there may be other factors such as disulfide bond geometry and access to oxidant which influence the rate of disulfide bond formation, the collision rate between the two thiols which is related to the interresidue distance is likely to play a principal role.…”

Section: Discussionmentioning

confidence: 99%

“…Purification via the C-terminal 1D4 epitope followed by Western blot detection of the N-terminal fragment ensures that only split receptors consisting of both N-and C-terminal fragments are probed. If a split receptor mutant has been cross-linked, then the protein migrates with a mobility similar to that of full-length, wild-type rhodopsin, while the mobility of a noncross-linked split receptor is the same as the isolated N-terminal fragment, SR (1)(2)(3)(4)(5).…”

Section: Methodsmentioning

confidence: 99%

“…The lack of precise crystallographic structural information for membrane proteins such as rhodopsin has prompted the development of a number of biochemical methods for investigating the structure of membrane proteins. One such method, which has been used to map out tertiary interactions in the homodimeric bacterial chemotatic receptors, is cysteine scanning mutagenesis and oxidative disulfide cross-linking (3)(4)(5)(6). We have recently adapted this method to monomeric GPCRs such as rhodopsin by utilizing split receptor (SR) constructs in which fully functional receptors are expressed as noncovalently associated N-and C-terminal fragments (7).…”

mentioning

confidence: 99%

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Tertiary Interactions between the Fifth and Sixth Transmembrane Segments of Rhodopsin

Struthers

Kono

et al. 1999

Biochemistry

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We have used cysteine scanning mutagenesis and disulfide cross-linking in a split rhodopsin construct to investigate the secondary structure and tertiary contacts of the fifth (TM5) and sixth (TM6) transmembrane segments of rhodopsin. Using a simple increase in pH to promote disulfide bond formation, three cross-links between residues on the extracellular side of TM5 (at positions 198, 200, and 204) and TM6 (at position 276) have been identified and characterized. The helical pattern of cross-linking observed indicates that the fifth transmembrane helix extends through residue 200 but does not include residue 198. Rhodopsin mutants containing these disulfides demonstrate nativelike absorption spectra and light-dependent activation of transducin, suggesting that large movements on the extracellular side of TM5 with respect to TM6 are not required for receptor activation.

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“…The structures and mechanisms of the transmembrane signaling domain, consisting of the periplasmic ligand binding domain and the membrane-spanning helices, are well characterized for the aspartate receptor and for the closely related serine and ribose/galactose chemotaxis receptors (22)(23)(24)(25)(26)(27)(28)(29). The architecture of the periplasmic and transmembrane regions consists of R-helical bundles oriented roughly perpendicular to the bilayer (22)(23)(24)30).…”

mentioning

confidence: 99%

Signaling Domain of the Aspartate Receptor Is a Helical Hairpin with a Localized Kinase Docking Surface: Cysteine and Disulfide Scanning Studies

1999

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Cysteine and disulfide scanning has been employed to probe the signaling domain, a highly conserved motif found in the cytoplasmic region of the aspartate receptor of bacterial chemotaxis and related members of the taxis receptor family. Previous work has characterized the N-terminal section of the signaling domain [Bass, R. B., and Falke, J. J. (1998) J. Biol. Chem. 273, 25006-25014], while the present study focuses on the C-terminal section and the interactions between these two regions. Engineered cysteine residues are incorporated at positions Gly388 through Ile419 in the signaling domain, thereby generating a library of receptors each containing a single cysteine per receptor subunit. The solvent exposure of each cysteine is ascertained by chemical reactivity measurements, revealing a periodic pattern of buried hydrophobic and exposed polar residues characteristic of an amphipathic α-helix, denoted helix α8. The helix begins between positions R392 and Val401, then continues through the last residue scanned, Ile419. Activity assays carried out both in vivo and in vitro indicate that both the buried and exposed faces of this amphipathic helix are critical for proper receptor function and the buried surface is especially important for kinase downregulation. Patterns of disulfide bond formation suggest that helix α8, together with the immediately N-terminal helix α7, forms a helical hairpin that associates with a symmetric hairpin from the other subunit of the homodimer, generating an antiparallel four helix bundle containing helices α7, α7′, α8, and α8′. Finally, the protein-interactions-by-cysteine-modification (PICM) method suggests that the loop between helices α7 and α8 interacts with the kinase CheA and/or the coupling protein CheW, expanding the receptor surface implicated in kinase docking.The transmembrane aspartate receptor of Escherichia coli and Salmonella typhimurium chemotaxis is a member of the large receptor superfamily that modulates two-component signaling systems in prokaryotes and eukaryotes (1-9). A subfamily of these receptors, the methyl-accepting taxis receptors, share the highest sequence homology with the aspartate receptor and function in chemo-, thermo-, osmo-, photo-, and pH-taxis in a wide range of prokaryotic organisms (5,6,10,11). The aspartate receptor is the best characterized member of this subfamily and has become a paradigm for two-component pathway receptors in general.The aspartate receptor exists as a stable homodimer in which each subunit is approximately 60 kDa as schematically illustrated in Figure 1. The receptor possesses a periplasmic ligand binding domain, a transmembrane region, and a cytoplasmic domain that possesses adaptation sites and a kinase docking surface. The transmembrane and adaptation signals relayed by the † Support provided by NIH Grant GM R01-40731 (to J.J.F.).

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Deducing the organization of a transmembrane domain by disulfide cross-linking. The bacterial chemoreceptor Trg.

Cited by 101 publications

References 29 publications

Transmembrane signaling by the aspartate receptor: Engineered disulfides reveal static regions of the subunit interface

Transmembrane signaling by the aspartate receptor: Engineered disulfides reveal static regions of the subunit interface

Tertiary Interactions between the Fifth and Sixth Transmembrane Segments of Rhodopsin

Signaling Domain of the Aspartate Receptor Is a Helical Hairpin with a Localized Kinase Docking Surface: Cysteine and Disulfide Scanning Studies

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