Amino-acid residues involved in the expression of the activity of Escherichia coli TolC.
The Escherichia coli TolC, composed of 471 amino-acid residues, functions as a channel tunnel in the transport of various molecules across the outer membrane. We found previously that Leu-412, the 60th amino-acid residue from the carboxy terminal end, was crucial to the transport activity of TolC. Leu-412 is located in a domain which protrudes from the main body of TolC into the periplasm. Subsequent study indicated that the hydrophobicity generated by Leu-412 played an important role in the activity of TolC (H. Yamanaka, T. Nomura, N. Morisada, S. Shinoda, and K. Okamoto, Microb. Pathog. 33: 81-89, 2002). We predicted that other hydrophobic amino-acid residues around Leu-412 were also involved in the expression of the activity of TolC. To test this possibility, we substituted several hydrophobic residues around Leu-412, (Leu-3, Val-6, Leu-212, Leu-213, Leu-223, and Leu-224), with serine and examined the activity of these mutant TolCs. The result showed that Leu-3 is involved in the activity of TolC, but the other residues are not. The involvement of Leu-3 was confirmed by the residue deletion experiment. A subsequent point-mutational analysis of the residue showed that a hydrophobic side chain is required at position 3 for TolC to express its activity. As the distance between the alpha-carbons of Leu-3 and Leu-412 is just 7.45 angstroms, hydrophobic interaction between the two leucine residues might be involved in the activity of TolC.
Yamanaka
Hiroyasu
H
Laboratory of Biochemistry, Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima, Japan. yama@ph.bunri-u.ac.jp
Morisada
Naoyuki
N
Miyano
Masaya
M
Tsuge
Hideaki
H
Shinoda
Sumio
S
Takahashi
Eizo
E
Okamoto
Keinosuke
K
D000596:Amino Acids / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003087:Colicins / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015870:Gene Expression / Q000502:physiology*; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D021381:Protein Transport
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli