title: Insights into the effect of detergents on the full-length rhomboid protease from Pseudomonas aeruginosa and its cytosolic domain.
abstract: Rhomboids comprise a family of intramembrane serine proteases that catalyze the cleavage of transmembrane segments within the lipid membrane to achieve a wide range of biological functions. A subset of bacterial rhomboids possesses an N-terminal cytosolic domain that appears to enhance proteolytic activity via an unknown mechanism. Structural analysis of a full-length rhomboid would provide new insights into this mechanism, an objective that solution NMR has the potential to realize. For this purpose we purified the rhomboid from Pseudomonas aeruginosa in a range of membrane-mimetic media, evaluated its functional status in vitro and investigated the NMR spectroscopic properties of these samples. In general, NMR signals could only be observed from the cytosolic domain, and only in detergents that did not support rhomboid activity. In contrast, media that supported rhomboid function did not show these resonances, suggesting an association between the cytosolic domain and the protein-detergent complex. Investigations into the ability of the isolated cytosolic domain to bind detergent micelles revealed a denaturing interaction, whereas no interaction occurred with micelles that supported rhomboid activity. The cytosolic domain also did not show any tendency to interact with lipid bilayers found in small bicelles or vesicles made from Escherichia coli phospholipid extracts. Based on these data we propose that the cytosolic domain does not interact with the lipid membrane, but instead enhances rhomboid activity through interactions with some other part of the rhomboid, such as the catalytic core domain.
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5.
authors.forename: Allison R
authors.initials: AR
authors.lastname: Sherratt
authors.forename: Michael V
authors.initials: MV
authors.lastname: Braganza
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Nguyen
authors.forename: Thierry
authors.initials: T
authors.lastname: Ducat
authors.forename: Natalie K
authors.initials: NK
authors.lastname: Goto
mesh_terms: D000595:Amino Acid Sequence; D003600:Cytosol / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology*; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008565:Membrane Proteins / Q000378:metabolism; D008566:Membranes / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000187:drug effects* / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D057057:Serine Proteases / Q000187:drug effects*
chemical_list: D004268:DNA-Binding Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; C102898:GlpG protein, E coli; D008565:Membrane Proteins; D010450:Endopeptidases; D057057:Serine Proteases