Lipid-layer crystallization and preliminary three-dimensional structural analysis of AcrA, the periplasmic component of a bacterial multidrug efflux pump.
The multidrug efflux complex AcrAB-TolC confers intrinsic drug resistance in Escherichia coli by pumping antibiotics out of the cell. We determined a low-resolution (20 A) structure of AcrA, the periplasmic component, by electron crystallography. Expressed with a His-tag at its carboxyl-terminus, the protein bound to lipid layers containing the nickel-chelating phospholipid DOGS-NTA. Under the lipid layers, AcrA crystallized in layer group P2(1)22, with a unit cell size of 157 by 95 A and a thickness of about 100 A. The four asymmetric units in the unit cell are organized into what appears to be two rings, each with a central opening of 30 A in diameter. Within each ring, the density can be interpreted as following a pseudo-helical path, approximately 210 A long. This length matches that of monomeric AcrA in solution, previously estimated by light scattering and hydrodynamic measurements. On one side the density has a tubular shape, with a thickness of about 25 A, while on the other side the densities of the upper and lower parts of the pseudo-helical path are fused into a shield.
Avila-Sakar
A J
AJ
Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Mailstop Donner, Berkeley, CA 94720, USA. AJAvila-Sakar@lbl.gov
Misaghi
S
S
Wilson-Kubalek
E M
EM
Downing
K H
KH
Zgurskaya
H
H
Nikaido
H
H
Nogales
E
E
D001426:Bacterial Proteins / Q000737:chemistry*; D002352:Carrier Proteins; D003461:Crystallography / Q000379:methods; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D005583:Fourier Analysis; D008074:Lipoproteins / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D026901:Membrane Transport Proteins; D008854:Microscopy, Electron / Q000379:methods; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D019897:Periplasm / Q000737:chemistry
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083497:AcrA protein, E coli; C083496:AcrB protein, E coli