Analysis of protein structure in intact cells: Crosslinking in vivo between introduced cysteines in the transmembrane domain of a bacterial chemoreceptor

Hughson, Andrew G.; Lee, G. F.; Hazelbauer, Gerald L.

doi:10.1002/pro.5560060206

Cited by 59 publications

(71 citation statements)

References 34 publications

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“…3). This indiscriminate cross-linking is reminiscent of findings with other protein complexes after prolonged exposure to CuP and has been attributed to rotational mobility or flexibility of the protein backbone (25,26). In contrast, the increase in cross-linking in the C-terminal half of TM1 was relatively selective for positions 98, 101, and 105, reinforcing the evidence of a helical repeat, and the fractional cross-linking at residues 99, 100, 103, and 104 remained very low (Fig.…”

Section: Resultsmentioning

confidence: 74%

Pore architecture of the ORAI1 store-operated calcium channel

Zhou

Ramachandran

Oh‐hora

et al. 2010

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

135

176

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ORAI1 is the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a store-operated channel that is central to Ca 2þ signaling in mammalian cells. Electrophysiological data have shown that the acidic residues E106 in transmembrane helix 1 (TM1) and E190 in TM3 contribute to the high selectivity of ORAI1 channels for Ca 2þ . We have examined the pore architecture of the ORAI1 channel using ORAI1 proteins engineered to contain either one or two cysteine residues. Disulfide cross-linking shows that ORAI1 assembles as a tetramer or a higher oligomer with TM1 centrally located. Cysteine side chains projecting from TM1 at position 88, 95, 102, or 106 cross-link efficiently to the corresponding side chain in a second ORAI1 monomer. Cysteine residues at position 190 or at surrounding positions in TM3 do not cross-link. We conclude that E106 residues in wild-type ORAI1 are positioned to form a Ca 2þ binding site in the channel pore and that E190 interacts less directly with ions traversing the pore. The cross-linking data further identify a relatively rigid segment of TM1 adjacent to E106 that is likely to contribute to the selectivity filter.cross-linking | membrane protein | store-operated calcium entry | stromal interaction molecule | structure T he electrophysiologically defined calcium release-activated calcium (CRAC) channel is responsible for physiological Ca 2þ influx in T cells and mast cells and for store-operated Ca 2þ entry in other cells (1). CRAC currents in human T cells require the protein ORAI1 (2-4), which has been shown to be a plasma membrane Ca 2þ channel protein [(5-8); reviewed in refs. 9-11]. The severe combined immunodeficiencies that result from rare mutations in the human ORAI1 gene and the functional deficits of Orai1 −∕− mice dramatize the crucial signaling role played by ORAI1 in Tcells and mast cells (2,(12)(13)(14). The hallmarks of native CRAC channels and of recombinant ORAI1 channels are that they open in response to a reduction of Ca 2þ concentration in the endoplasmic reticulum (ER) lumen, that they are highly selective for Ca 2þ under physiological conditions, and that they carry only very small single-channel currents (1, 11). The sensitivity of the channel to the level of ER Ca 2þ stores has been traced to the ER-resident Ca 2þ sensor protein STIM1 (15)(16)(17)(18)(19)(20), but the basis for other channel properties is not understood in any detail.Point mutations introduced into the transmembrane helices of ORAI1 or its Drosophila orthologue alter ion selectivity (5-7). The replacements E106D or E190Q in human ORAI1 sharply reduce the ability of the channel to discriminate between Ca 2þ and Na þ under physiological conditions and reduce the decided preference of the channel for Na þ over Cs þ under conditions where the channel conducts monovalent ions (6, 7). However, although the electrophysiological analysis shows that E106 and E190 influence ion movements within the channel pore, it does not establish whether these residues do so directly by coordinating ...

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Section: Resultsmentioning

confidence: 74%

Pore architecture of the ORAI1 store-operated calcium channel

Zhou

Ramachandran

Oh‐hora

et al. 2010

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

135

176

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“…The mechanism of transmembrane signaling is a subtle piston-type displacement of the signaling helix ␣4/TM2 toward the cytoplasm, as observed in the superimposed crystal structures of the apo-and aspartate-occupied periplasmic domain (43). Further evidence for the piston displacement is provided by engineered disulfide bonds that lock the membrane-bound receptor in the on and off states, by ligand-induced changes in disulfide formation rates detected in a closely related receptor for ribose and galactose, and, most recently, by spin-labeled ESR measurements (34,39,(43)(44)(45).…”

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confidence: 83%

Detection of a Conserved α-Helix in the Kinase-docking Region of the Aspartate Receptor by Cysteine and Disulfide Scanning

Bass

Falke

1998

Journal of Biological Chemistry

139

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The transmembrane aspartate receptor of Escherichia coli and Salmonella typhimurium propagates extracellular signals to the cytoplasm, where its cytoplasmic domain regulates the histidine kinase, CheA. Different signaling states of the cytoplasmic domain modulate the kinase autophosphorylation rate over at least a 100-fold range. Biochemical and genetic studies have implicated a specific region of the cytoplasmic domain, termed the signaling subdomain, as the region that transmits regulation from the receptor to the kinase. Here cysteine and disulfide scanning are applied to the N-terminal half of the signaling subdomain to probe its secondary structure, solvent exposure, and protein-protein interactions. The chemical reactivities of the scanned cysteines exhibit the characteristic periodicity of an ␣-helix with distinct solvent-exposed and buried faces. This helix, termed ␣7, ranges approximately from residue 355 through 386. Activity measurements probing the effects of cysteine substitutions in vivo and in vitro reveal that both faces of helix ␣7 are critical for kinase activation, while the buried face is especially critical for kinase down-regulation. Disulfide scanning of the region suggests that helix ␣7 is not in direct contact with its symmetric partner (␣7) from the other subunit; presently, the structural element that packs against the buried face of the helix remains unidentified. Finally, a novel approach termed "protein interactions by cysteine modification" indicates that the exposed C-terminal face of helix ␣7 provides an essential docking site for the kinase CheA or for the coupling protein CheW.A fundamental question in signaling biology concerns the mechanism by which cell surface receptors regulate cytoplasmic kinases. Certain receptors activate their associated kinases by dimerization, but in other cases kinase activation is triggered by a transmembrane conformational change. One class of receptors that generates such conformational regulation is the large group of cell surface receptors that modulate histidine kinases in prokaryotic and eukaryotic two-component signaling pathways (reviewed in Refs. 1-9). The aspartate receptor of bacterial chemotaxis is representative of this diverse class. The aspartate receptor is one of the ligand specific dimeric receptors utilized by the related chemotaxis pathways of Escherichia coli and Salmonella typhimurium to recognize periplasmic attractant and repellent molecules. This receptor binds aspartate, an attractant, in the periplasm and propagates a signal across the bilayer to an associated histidine kinase in the cytoplasm. Receptor-kinase coupling is provided by the formation of a kinetically stable ternary complex between the cytoplasmic domain of the receptor, the coupling protein CheW, and the histidine kinase CheA (10, 11). The cytoplasmic phosphorylation cascade is initiated by receptorstimulated CheA autophosphorylation; subsequently, phosphoCheA becomes the substrate for a phospho-transfer reaction that phosphorylates one of two response regul...

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“…Attractant binding to the receptor generates a piston (or swinging-piston) displacement of the signaling helix toward the cytoplasm (40). Parallel studies have shown that the related chemoreceptor for ribose and galactose shares the same helical structure and piston-type signaling mechanism, illustrating the generality of this transmembrane signaling motif at least within the bacterial chemoreceptor subfamily of two-transmembrane helix receptors (41)(42)(43)(44)(45)(46).…”

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confidence: 98%

Cysteine and Disulfide Scanning Reveals a Regulatory α-Helix in the Cytoplasmic Domain of the Aspartate Receptor

Danielson

Bass

Falke

1997

Journal of Biological Chemistry

186

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The transmembrane, homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium controls the chemotactic response to aspartate, an attractant, by regulating the activity of a cytoplasmic histidine kinase. The cytoplasmic domain of the receptor plays a central role in both kinase regulation and sensory adaptation, although its structure and regulatory mechanisms are unknown. The present study utilizes cysteine and disulfide scanning to probe residues Leu-250 through Gln-309, a region that contains the first of two adaptive methylation segments within the cytoplasmic domain. Following the introduction of consecutive cysteine residues by scanning mutagenesis, the measurement of sulfhydryl chemical reactivities reveals an ␣-helical pattern of exposed and buried positions spanning residues 270 -309. This detected helix, termed the "first methylation helix," is strongly amphiphilic; its exposed face is highly anionic and possesses three methylation sites, while its buried face is hydrophobic. In vivo and in vitro assays of receptor function indicate that inhibitory cysteine substitutions are most prevalent on the buried face of the first methylation helix, demonstrating that this face is involved in a critical packing interaction. The buried face is further analyzed by disulfide scanning, which reveals three "lock-on" disulfides that covalently trap the receptor in its kinaseactivating state. Each of the lock-on disulfides crosslinks the buried faces of the two symmetric first methylation helices of the dimer, placing these helices in direct contact at the subunit interface. Comparative sequence analysis of 56 related receptors suggests that the identified helix is a conserved feature of this large receptor family, wherein it is likely to play a general role in adaptation and kinase regulation. Interestingly, the rapid rates and promiscuous nature of disulfide formation reactions within the scanned region reveal that the cytoplasmic domain of the full-length, membranebound receptor has a highly dynamic structure. Overall, the results demonstrate that cysteine and disulfide scanning can identify secondary structure elements and functionally important packing interfaces, even in proteins that are inaccessible to other structural methods.The aspartate receptor of Escherichia coli and Salmonella typhimurium is representative of a large family of cell-surface receptors that regulate two-component signaling pathways, which are widespread in prokaryotic and eukaryotic organisms (1-9). These receptors contain two putative transmembrane helices per subunit and, in all cases tested, form stable homodimers that signal via a transmembrane conformational change. Chimeric receptors containing the sensory domain of the aspartate receptor and the regulatory domain of another family member are functional, suggesting that members of this receptor family use a conserved mechanism of transmembrane signaling to regulate cytoplasmic histidine kinase activity (10 -12). More generally, conformational transmembrane signals ma...

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Analysis of protein structure in intact cells: Crosslinking in vivo between introduced cysteines in the transmembrane domain of a bacterial chemoreceptor

Cited by 59 publications

References 34 publications

Pore architecture of the ORAI1 store-operated calcium channel

Pore architecture of the ORAI1 store-operated calcium channel

Detection of a Conserved α-Helix in the Kinase-docking Region of the Aspartate Receptor by Cysteine and Disulfide Scanning

Cysteine and Disulfide Scanning Reveals a Regulatory α-Helix in the Cytoplasmic Domain of the Aspartate Receptor

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