The structure of the efflux pump AcrB in complex with bile acid

Figures & data

Table I.  Data collection and refinement statistics.

Data collection	Deoxycholate	Native
Space group	H32
Beamline	Diamond I04	ESRF ID 14 4
Cell dimensions
a, b, c (Å)	145.49, 145.49, 515.18	146.29, 146.29, 513.7
α, β, γ (°)		90.0, 90.0, 120.0
Resolution (Å)	3.85 (3.85)	3.2 (3.2)
Rsym or Rmerge^#	23.4 (56.4)	7.6 (52.2)
I/σ	2.5 (1.3)	8.3 (1.4)
Completeness (%)	96.4 (96.2)	98.8 (100)
Redundancy	4.1 (4.2)	3.5 (3.5)
Refinement
Resolution (Å)	3.85	3.2
No. reflections	18558	32895
Rwork/Rfree *	27.7/34.9	30.98/36.5
No. atoms
Protein	7841
Ligand	28
Average B-factors (A^2)	75	100
R.m.s. deviations
Bond lengths (Å)	0.012	0.01
Bond angles (°)	1.39	1.3

Values in parentheses are for the high-resolution shells; ^#R_merge=Σ_hkl Σ_I ∣I_i(hkl)- < I(hkl)>∣/Σ_hkl Σ_I I_i(hkl); ^*R_factor and R_free =Σ_hkl [∣F_o(hkl)∣-∣F_c(hkl)∣]/ Σ_hkl ∣F_o(hkl)∣; R_free was calculated using 5% of the data.

Figure 1.  Comparison of growth of an E. coli wild-type strain with an acrB deletion strain in the presence of deoxycholate (DOC). thin line, BL21(DE3) in absence of bile acid; thin dashed line, BL21(DE3) with 5 mM addition of deoxycholate; thick line, BL21(DE3)ΔacrB::Km^R in absence of bile acid, thick dashed-line, BL21(DE3)ΔacrB::Km^R with 5 mM addition of deoxycholate.

Figure 2.  Fractions of a standard membrane protein-GFP-His₈ purification were loaded onto a 12% SDS-polyacrylamide gel and then stained with Coomassie to illustrate isolation of native AcrB. (1) MP-GFP-His₈ elution from first IMAC; (2) overnight His₆-TEV protease digested material; (3) flow-through of second IMAC step; and (4) elution of bound material from second IMAC (bands corresponding to AcrB, MP-GFP-His₈, His₆-TEVp, and MP are as indicted on gel).

Figure 3.  Overall structure of AcrB trimer. The bound deoxycholate in the periplasmic vestibule is shown in yellow stick. Boxed area is a close-up view of the vestibule site with the deoxycholate and side chains that are involved in the binding shown in grey stick.

Figure 4.  Binding of deoxycholate in the periplasmic vestibule of AcrB. (a) Electron density for native AcrB (purple), (b) deoxycholate bound prior to adding the ligand (light blue), (c) deoxycholate density after including it in the refinement (dark blue). The 2F_o-F_c maps are contoured at 1.0σ and deoxycholate (pink, contoured at 1.0σ) are shown for clarity, calculated after some model building (prior to deoxycholate being built). Some of the vestibule amino acid side chains are shown. Deoxycholate (yellow sticks) is included for reference. Nitrogen atoms are shown in blue and oxygen in red.

Figure 5.  Alignment of deoxycholate (yellow) with ethidium (light orange), Phe-Arg-β-naphthylamide (light blue), and ciprofloxacin (pink). The residues involved in the binding of deoxycholate are shown as grey sticks. Arg⁷¹⁷ is only shown as a reference since there is no direct contact with the deoxycholate.