Proximity Mapping the Surface of a Membrane Protein Using an Artificial Protease: Demonstration That the Quinone-Binding Domain of Subunit I Is near the N-Terminal Region of Subunit II of Cytochrome bd

Ghaim, Joshua B.; Greiner, Douglas P.; Meares, Claude F.; Gennis, Robert B.

doi:10.1021/bi00036a002

Cited by 57 publications

(39 citation statements)

References 44 publications

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“…Alternatively, short living hydroxyl radicals can abstract a hydrogen from the ␣-carbon of an amino acid leading to an unstable carbon-centered radical that degrades, forming new blocked termini (18). In both cases, the cleavage is limited to a 1.2-nm distance from the attachment site of the chemical protease (29,30). The cleavage is very fast and achieved within 10 s (see Fig.…”

Section: Discussionmentioning

confidence: 99%

Mechanism of Substrate Sensing and Signal Transmission within an ABC Transporter

Herget

Oancea

Schrodt

et al. 2007

Journal of Biological Chemistry

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By translocating proteasomal degradation products into the endoplasmic reticulum for loading of major histocompatibility complex I molecules, the ABC transporter TAP plays a focal role in the adaptive immunity against infected or malignantly transformed cells. A key question regarding the transport mechanism is how the quality of the incoming peptide is detected and how this information is transmitted to the ATPase domains. To identify residues involved in this process, we evolved a Trojan horse strategy in which a small artificial protease is inserted into antigenic epitopes. After binding, the TAP backbone in contact is cleaved, allowing the peptide sensor site to be mapped by mass spectrometry. Within this sensor site, we identified residues that are essential for tight coupling of peptide binding and transport. This sensor and transmission interface is restructured during the ATP hydrolysis cycle, emphasizing its important function in the cross-talk between the transmembrane and the nucleotidebinding domains. This allocrite sensor may be similarly positioned in other members of the ABC exporter family.The transporter associated with antigen processing (TAP) 4 plays a prominent role in the antigen-processing pathway via major histocompatibility complex (MHC) class I molecules (1, 2). A fraction of proteasomal degradation products is translocated into the endoplasmic reticulum by the TAP complex and loaded onto MHC class I molecules. Peptide-loaded MHC complexes can pass the endoplasmic reticulum quality control and traffic to the cell surface, where they display their antigenic cargo to cytotoxic T-lymphocytes, which can thereby efficiently recognize and eliminate infected or malignantly transformed cells. TAP belongs to the superfamily of ATP-binding cassette (ABC) transporters, which translocate a very broad spectrum of substrates across membranes (3, 4).The TAP complex forms a TAP1/TAP2 heterodimer; each subunit contains a transmembrane domain (TMD) followed by a cytosolic nucleotide-binding domain (NBD). The translocation mechanism can be dissected into an ATP-independent peptide binding and an ATP-dependent translocation step (5). TAP1 and TAP2 are required and sufficient for both processes (5, 6). Peptide binding is composed of a fast association step followed by slow structural reorganization of the transport complex (7,8). Previous studies demonstrated that peptide binding to the TMDs triggers ATP hydrolysis by the NBDs (9, 10). TAP preferentially binds peptides with a length of 8 -16 amino acids (5). Using combinatorial peptide libraries, the first three N-terminal and the last C-terminal residues were identified as critical for peptide binding, whereas amino acids between these "anchor" positions do not significantly contribute to the substrate recognition (11). Remarkably, TAP can also bind peptides with large bulky side chains, including cross-linkers, fluorophors, or extended side chains (9, 12, 13). Strikingly, however, sterically restricted peptides that bind but are not transported do not stim...

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Section: Discussionmentioning

confidence: 99%

Mechanism of Substrate Sensing and Signal Transmission within an ABC Transporter

Herget

Oancea

Schrodt

et al. 2007

Journal of Biological Chemistry

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“…The different mutations observed indicate that the different isolates were, for the most part, the result of independent events. All but two of the observed mutations in the cydA product appeared to be located in the portion of the protein found in the periplasmic space (16,17,22), and considering the stop codons observed, it is likely that the mutations inactivated the enzyme. The wildtype cydA ϩ gene was able to complement the suppressed strain to restore filamentous killing.…”

Section: ϫ6mentioning

confidence: 97%

Cell Death in Escherichia coli dnaE (Ts) Mutants Incubated at a Nonpermissive Temperature Is Prevented by Mutation in the cydA Gene

et al. 2004

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Cells of the Escherichia coli dnaE(Ts) dnaE74 and dnaE486 mutants die after 4 h of incubation at 40°C in Luria-Bertani medium. Cell death is preceded by elongation, is inhibited by chloramphenicol, tetracycline, or rifampin, and is dependent on cell density. Cells survive at 40°C when they are incubated at a high population density or at a low density in conditioned medium, but they die when the medium is supplemented with glucose and amino acids. Deletion of recA or sulA has no effect. We isolated suppressors which survived for long periods at 40°C but did not form colonies. The suppressors protected against hydroxyurea-induced killing. Sequence and complementation analysis indicated that suppression was due to mutation in the cydA gene. The DNA content of dnaE mutants increased about eightfold in 4 h at 40°C, as did the DNA content of the suppressed strains. The amount of plasmid pBR322 in a dnaE74 strain increased about fourfold, as measured on gels, and the electrophoretic pattern appeared to be normal even though the viability of the parent cells decreased 2 logs. Transformation activity also increased. 4,6-Diamidino-2-phenylindole staining demonstrated that there were nucleoids distributed throughout the dnaE filaments formed at 40°C, indicating that there was segregation of the newly formed DNA. We concluded that the DNA synthesized was physiologically competent, particularly since the number of viable cells of the suppressed strain increased during the first few hours of incubation. These observations support the view that E. coli senses the rate of DNA synthesis and inhibits septation when the rate of DNA synthesis falls below a critical level relative to the level of RNA and protein synthesis.Cell division and DNA synthesis are coupled in all organisms, but the mechanism of the coupling is not clear, notwithstanding the sophisticated description of an operon with multiple promoters determining the production of the FtsZ and other proteins involved in cell division (12,15). In Escherichia coli, the completion of a round of DNA synthesis is usually followed by activation of the cell septation machinery, but synthesis does not trigger septation (4). DNA damage or other blocks to DNA synthesis often lead to inhibition of cell division. For example, induction of the SOS repair pathway activates the sulA gene product, inhibiting cell division (5). Some signal recognizes that the DNA has been damaged (or that synthesis has been inhibited), and as a result division is suspended. Deprivation of thymine necessarily inhibits DNA synthesis and also leads to filamentation (1). Filamentation often accompanies cell death, and there have been numerous studies attempting to elucidate the mechanism of death both as a result of thymine starvation and as a result of other pathways to filamentation.As part of an investigation of the roles of the different DNA polymerases in mutation (35), we studied the behavior of strains carrying a temperature-sensitive (Ts) mutation in the alpha subunit of the replicative DNA polymerase...

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“…The former group includes most of the c-proteobacteria, while the latter group includes all of the Gram-positive bacteria, the cyanobacterium, and the c-proteobacterium Pseudomonas aeruginosa (Osborne and Gennis 1999;Sakamoto et al 1999). It has been demonstrated that the Q-loop is adjacent to loop I-II in subunit II (Ghaim et al 1995). Furthermore, the Q-loop is known to be involved in quinol binding (Ghaim et al 1995).…”

Section: Introductionmentioning

confidence: 99%

“…It has been demonstrated that the Q-loop is adjacent to loop I-II in subunit II (Ghaim et al 1995). Furthermore, the Q-loop is known to be involved in quinol binding (Ghaim et al 1995). The cytochrome bd complex contains three prosthetic groups: heme b 558 , heme b 595 , and heme d. Heme b 558 and heme b 595 are protoporphyrin, while heme d is a chlorin (Timkovich et al 1985).…”

Section: Introductionmentioning

confidence: 99%

Asymmetrical Evolution of Cytochrome bd Subunits

Hao

Golding

2006

J Mol Evol

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Abstract. Functionally linked genes generally evolve at similar rates and the knowledge of this particular feature of genomic evolution has been used as the basis for the phylogenetic profiling method. We illustrate here an exception to this rule in the evolution of the cytochrome bd complex. This is a twocomponent oxidase complex, with the subunits I and II known to be widely present in bacteria. The subunits within the cytochrome bd complex are under the same evolutionary pressure and most likely behave in the same evolutionary manner. However, the sequence similarity of genes encoding subunit II varies considerably across species. Genes encoding subunit II evolve 1.2 times faster on most of the branches of their phylogeny than subunit I genes. Furthermore, the genes encoding subunit II in Oceanobacillus iheyensis, Bacillus halodurans, and Staphylococcus species do not have detectable homologues within E. coli due to their large divergence. Together, the two subunits of cytochrome bd reveal an interesting example of an asymmetric pattern of evolutionary change.

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Proximity Mapping the Surface of a Membrane Protein Using an Artificial Protease: Demonstration That the Quinone-Binding Domain of Subunit I Is near the N-Terminal Region of Subunit II of Cytochrome bd

Cited by 57 publications

References 44 publications

Mechanism of Substrate Sensing and Signal Transmission within an ABC Transporter

Mechanism of Substrate Sensing and Signal Transmission within an ABC Transporter

Cell Death in Escherichia coli dnaE (Ts) Mutants Incubated at a Nonpermissive Temperature Is Prevented by Mutation in the cydA Gene

Asymmetrical Evolution of Cytochrome bd Subunits

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