Structure and dynamics of the membrane-embedded domain of LmrAinvestigated by coupling polarized ATR-FTIR spectroscopy and (1)H/(2)H exchange.
Bacterial LmrA, an integral membrane protein of Lactococcus lactis, confers multidrug resistance by mediating active extrusion of a wide variety of structurally unrelated compounds. Similar to its eucaryotic homologue P-gp, this protein is a member of the ATP-binding cassette (ABC) superfamily. Different predictive models, based on hydropathy profiles, have been proposed to describe the structure of the ABC transporters in general and of LmrA in particular. We used polarized attenuated total reflection infrared spectroscopy, combined with limited proteolysis, to investigate the secondary structure and the orientation of the transmembrane segments of LmrA. We bring the first experimental evidence that the membrane-embedded domain of LmrA is composed of transmembrane-oriented alpha-helices. Furthermore, a new approach was developed in order to provide information about membrane domain dynamics. Monitoring the infrared linear dichroism spectra in the course of (1)H/(2)H exchange allowed to focus the recording of exchange rates on the membrane-embedded region of the protein only. This approach revealed an unusual structural dynamics, indicating high flexibility in this antibiotic binding and transport region.
Grimard
V
V
Laboratory of Structure and Function of Biological Membranes (SFMB), Free University of Brussels, B-1050 Brussels, Belgium.
Vigano
C
C
Margolles
A
A
Wattiez
R
R
van Veen
H W
HW
Konings
W N
WN
Ruysschaert
J M
JM
Goormaghtigh
E
E
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006859:Hydrogen; D006868:Hydrolysis; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013329:Structure-Activity Relationship
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D006859:Hydrogen; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C101824:LmrA protein, Lactococcus lactis