The aspartate receptor cytoplasmic domain: in situ chemical analysis of structure, mechanism and dynamics.

BACKGROUND: Site-directed sulfhydryl chemistry and spectroscopy can be used to probe protein structure, mechanism and dynamics in situ. The aspartate receptor of bacterial chemotaxis is representative of a large family of prokaryotic and eukaryotic receptors that regulate histidine kinases in two-component signaling pathways, and has become one of the best characterized transmembrane receptors. We report here the use of cysteine and disulfide scanning to probe the helix-packing architecture of the cytoplasmic domain of the aspartate receptor. RESULTS: A series of designed cysteine pairs have been used to detect proximities between cytoplasmic helices in the full-length, membrane-bound receptor by measurement of disulfide-bond formation rates. Upon mild oxidation, 25 disulfide bonds from rapidly between three specific pairs of helices, whereas other helix pairs yield no detectable disulfide-bond formation. Further constraints on helix packing are provided by 14 disulfide bonds that retain receptor function in an in vitro kinase regulation assay. Of these functional disulfides, seven lock the receptor in the conformation that constitutively stimulates kinase activity ('lock-on'), whereas the remaining seven retain normal kinase regulation. Finally, disulfide-trapping experiments in the absence of bound kinase reveal large-amplitude relative motions of adjacent helices, including helix translations and rotations of up to 19 A and 180 degrees, respectively. CONCLUSIONS: The 25 rapidly formed and 14 functional disulfide bonds identify helix-helix contacts and their register in the full-length, membrane-bound receptor-kinase complex. The results reveal an extended, rather than compact, domain architecture in which the observed helix-helix interactions are best described by a four-helix bundle arrangement. A cluster of six lock-on disulfide bonds pinpoints a region of the four-helix bundle critical for kinase activation, whereas the signal-retaining disulfides indicate that signal-induced rearrangements of this region are small enough to be accommodated by disulfide-bond flexibility (< or = 1.2 A). In the absence of bound kinase, helix packing within the cytoplasmic domain is highly dynamic.

Signaling domain of the aspartate receptor is a helical hairpin with a localized kinase docking surface: cysteine and disulfide scanning studies.

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 alpha-helix, denoted helix alpha8. 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 alpha8, together with the immediately N-terminal helix alpha7, 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 alpha7, alpha7', alpha8, and alpha8'. Finally, the protein-interactions-by-cysteine-modification (PICM) method suggests that the loop between helices alpha7 and alpha8 interacts with the kinase CheA and/or the coupling protein CheW, expanding the receptor surface implicated in kinase docking.

Detection of a conserved alpha-helix in the kinase-docking region of the aspartate receptor by cysteine and disulfide scanning.

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 alpha-helix with distinct solvent-exposed and buried faces. This helix, termed alpha7, 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 alpha7 are critical for kinase activation, while the buried face is especially critical for kinase down-regulation. Disulfide scanning of the region suggests that helix alpha7 is not in direct contact with its symmetric partner (alpha7') 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 alpha7 provides an essential docking site for the kinase CheA or for the coupling protein CheW.

The two-component signaling pathway of bacterial chemotaxis: a molecular view of signal transduction by receptors, kinases, and adaptation enzymes.

The chemosensory pathway of bacterial chemotaxis has become a paradigm for the two-component superfamily of receptor-regulated phosphorylation pathways. This simple pathway illustrates many of the fundamental principles and unanswered questions in the field of signaling biology. A molecular description of pathway function has progressed rapidly because it is accessible to diverse structural, biochemical, and genetic approaches. As a result, structures are emerging for most of the pathway elements, biochemical studies are elucidating the mechanisms of key signaling events, and genetic methods are revealing the intermolecular interactions that transmit information between components. Recent advances include (a) the first molecular picture of a conformational transmembrane signal in a cell surface receptor, (b) four new structures of kinase domains and adaptation enzymes, and (c) significant new insights into the mechanisms of receptor-mediated kinase regulation, receptor adaptation, and the phospho-activation of signaling proteins. Overall, the chemosensory pathway and the propulsion system it regulates provide an ideal system in which to probe molecular principles underlying complex cellular signaling and behavior.

Cysteine and disulfide scanning reveals a regulatory alpha-helix in the cytoplasmic domain of the aspartate receptor.

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 alpha-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 kinase-activating state. Each of the lock-on disulfides cross-links 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, membrane-bound 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 Medstone STS: a technical review.

The Medstone STS does not require the urologist to compromise his lithotripsy success because of insufficient power, inadequate imaging, or restrictions caused by patient positioning. The attention to the details required for success in treatment in the investigators opinion make the Medstone STS a very attractive device for the performance of lithotripsy.

Clinical application of transrectal ultrasonography and prostate specific antigen in the search for prostate cancer.

We reviewed 225 men who were followed for 2 to 21 years by periodic rectal examination in an effort to detect prostatic cancer without the glands having been sufficiently suspicious for biopsy to have been recommended. These patients underwent further evaluation with transrectal prostatic ultrasonography and serum prostate specific antigen determinations. When appropriate, ultrasonically guided transrectal needle biopsy of the prostate was performed without analgesia, anesthesia or prophylactic antibiotics. This is a simple, safe and effective means to obtain tissue for diagnosis. Recommendations for the current applicability of these diagnostic modalities by the practicing urologist are given.

Percutaneous ultrasonic lithotripsy in the community hospital.

A total of 38 patients underwent attempted percutaneous extraction of upper tract calculi with the Wolf nephroscope. A 1-day interval between radiological insertion of the guide wire and extraction of the stone with the patient under general anesthesia was used. Of the stones 87 per cent were removed successfully. Average operating and hospitalization times were 62 minutes and 9.9 days, respectively. Reduced morbidity and a shorter interval to return to work make this the procedure of choice in selected cases.

Radical retropubic prostatectomy after transurethral prostatic resection.

The 36 patients who underwent radical retropubic prostatectomy for adenocarcinoma after transurethral resection of the prostate were reviewed to evaluate the difficulty of the radical operation, the rate of morbidity and the survival. In 24 patients (67 per cent) the dissection proved to be routine; 18 patients (50 per cent) had perfect urinary control, whereas 4 were totally incontinent. There appears to be no untoward effect on survival, since all patients remain alive, 22 per cent having had followup for 5 years or more.