title: Evidence that the C-terminal region is involved in the stability and functionality of OprM in E. coli.
abstract: In order to understand the specificity of interactions between the components of multidrug-resistant (MDR) efflux pumps and how they are recruited/assembled, we analyzed the effect of C-terminal truncation, deletion, and peptide swapping on the stability and functionality of OprM in Escherichia coli. The efflux activity of OprM was not affected by removing up to 19 amino acid residues from the C-terminus, while depletion of more than 20 residues or disruption the ₄₆₃LGGG₄₆₆ motif diminished both the stability and activity of OprM. The replacement of the OprM C-terminus 23 residues with the corresponding part of TolC or VceC did not affect the stability and the functionality of OprM. Therefore, it is confirmed that the C-terminal ₄₆₃LGGG₄₆₆ motif is one of the crucial components for the stability of OprM and for the functionality of the OprM-VceAB chimeric pump in E.coli. The results also indicate that one residue substitution on the hairpin domain of the membrane fusion protein (MFP) VceA could suppress the null like mutations on the C-terminal modified OprM. This finding will be the direct genetic evidence that the C-terminal domain of outer efflux protein (OEP) is involved in the functional assembly of OEP-MFP.
authors.affiliation: Gansu Key Laboratory of Crop Improvement and Germplasm Enhancement, Key Lab of Aridland Crop Science, Gansu Agricultural University, Lanzhou, Gansu, 730070, China; Department of Microbiology and Immunology, Texas Tech University Health Science Center, Lubbock, TX 79410, USA. Electronic address: baijp@gsau.edu.cn.
authors.forename: Jiangping
authors.initials: J
authors.lastname: Bai
authors.affiliation: Department of Microbiology and Immunology, Texas Tech University Health Science Center, Lubbock, TX 79410, USA.
authors.forename: Ramasubramanian
authors.initials: R
authors.lastname: Bhagavathi
authors.affiliation: Department of Microbiology and Immunology, Texas Tech University Health Science Center, Lubbock, TX 79410, USA.
authors.forename: Phat
authors.initials: P
authors.lastname: Tran
authors.affiliation: JS Biosciences, Anning District, Lanzhou, Gansu, 730070, China.
authors.forename: Kendall
authors.initials: K
authors.lastname: Muzzarelli
authors.affiliation: Gansu Key Laboratory of Crop Improvement and Germplasm Enhancement, Key Lab of Aridland Crop Science, Gansu Agricultural University, Lanzhou, Gansu, 730070, China; Department of Microbiology and Immunology, Texas Tech University Health Science Center, Lubbock, TX 79410, USA.
authors.forename: Di
authors.initials: D
authors.lastname: Wang
authors.affiliation: Department of Microbiology and Immunology, Texas Tech University Health Science Center, Lubbock, TX 79410, USA.
authors.forename: Joe A
authors.initials: JA
authors.lastname: Fralick
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D055550:Protein Stability
chemical_list: D001425:Bacterial Outer Membrane Proteins; D026901:Membrane Transport Proteins; C402263:OprM protein, Pseudomonas aeruginosa
keywords: C-terminal domain; Gain-of-function mutations; Multidrug-resistant efflux pumps; OprM