title: Electrostatic Repulsion between Unique Arginine Residues Is Essential for the Efficient in Vitro Assembly of the Transmembrane Domain of a Trimeric Autotransporter.
abstract: Haemophilus influenzae adhesin (Hia) belongs to the trimeric autotransporter family and mediates the adherence of these bacteria to the epithelial cells of host organisms. Hia contains a passenger and a transmembrane domain. The transmembrane domain forms a 12-stranded β-barrel in which four strands are provided by each subunit. The β-barrel has a pore that is traversed by three α-helices. This domain has a unique arginine cluster, in which the side chains of the three arginine residues located at position 1077 (Arg1077) protrude into the pore of the β-barrel. This arrangement seems to be unfavorable for assembly, because of repulsion between the positive charges. In this study, we investigated the in vitro assembly of the Hia transmembrane minimum domain (mHiaTD) and found that the dissociated mHiaTD reassembled in detergent solution. To investigate the role of Arg1077 in trimer assembly, we generated mutant proteins in which Arg1077 was replaced with methionine or lysine. The reassembly kinetics of the mutants was compared with that of the wild-type protein. The methionine mutant showed misassembly, whereas the lysine mutant showed reversible assembly, similar to that observed for the wild-type protein. These results show that electrostatic repulsion between the positive charges of Arg1077 is important for preventing the formation of misassembled oligomers by the mHiaTD in vitro.
authors.affiliation: Department of Bioinformatics, Soka University , 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan.
authors.forename: Eriko
authors.initials: E
authors.lastname: Aoki
authors.affiliation: Department of Bioinformatics, Soka University , 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Sato
authors.affiliation: Department of Bioinformatics, Soka University , 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Fujiwara
authors.affiliation: Department of Bioinformatics, Soka University , 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan.
authors.forename: Masamichi
authors.identifier: 0000-0003-1190-1130
authors.initials: M
authors.lastname: Ikeguchi
mesh_terms: D001120:Arginine / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001704:Biopolymers / Q000737:chemistry*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D055672:Static Electricity
chemical_list: D001426:Bacterial Proteins; D001704:Biopolymers; D002352:Carrier Proteins; D008565:Membrane Proteins; D001120:Arginine