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Structural Basis of Substrate Recognition by the Multidrug Resistance Protein MRP1
Zachary Lee Johnson, Jue Chen Cell Volume 168, Issue 6, Pages e9 (March 2017) DOI: /j.cell Copyright © 2017 Elsevier Inc. Terms and Conditions
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Cell , e9DOI: ( /j.cell ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 1 Functional Characterization of Bovine MRP1
(A) ATPase activity of the full-length protein purified in digitonin. By nonlinear regression of the Michaelis-Menten equation, the KM for ATP was determined to be 400 ± 60 μM and the maximal ATPase activity was determined to be 27 ± 1 nmol/mg/min (assuming a pre-glycosylation molecular weight of 172 kDa, the specific ATP turnover rate is 4.7 min−1). (B and C) ATPase activity of the full-length MRP1 as a function of GSH (A) and LTC4 (B) concentrations. The protein and ATP concentrations were kept at 800 nM and 4 mM, respectively, for all measurements. The dashed lines represent the ATPase activity at 4 mM ATP in the absence of substrate. The KM value determined for GSH was 0.9 ± 0.2 mM and for LTC4 was 350 ± 90 nM. (D) ATPase activity of the truncation mutant devoid of TMD0. The KM for ATP was determined to be 240 ± 30 μM and the maximal ATPase activity was 28 ± 1 nmol/mg/min (or 4.2 min−1, assuming a molecular weight of 148 kDa). (E) At saturating concentrations (6 mM GSH or 20 μM LTC4), substrates stimulate the ATPase activity of the full-length MRP1 and the truncation mutant to similar levels. All measurements were carried out at 28°C. The reported errors represent SD. Cell , e9DOI: ( /j.cell ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 2 Quality of the Cryo-EM Data
(A and B) Drift-corrected micrographs at defocus of −2.4 μm and representative 2D class averages for the apo (A) and LTC4-bound (B) datasets. (C) Representative EM densities of transmembrane helices from the LTC4-bound map. See also Figures S1–S4. Cell , e9DOI: ( /j.cell ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 3 The Molecular Architecture of Apo MRP1
(A) Schematic of the domain structure of MRP1. Dashed lines represent regions not resolved in the cryo-EM reconstructions and not included in the models. The lasso motif is also known as the L0 linker. (B) Cartoon representation of bMRP1 viewed from within the plane of the membrane. Regions not included in the model are represented by dashed lines. (C) Cartoon representation of the transmembrane region viewed from the cytoplasm along the membrane normal. The shaded regions specify the two TM bundles discussed in the text. See also Figures S2–S4. Cell , e9DOI: ( /j.cell ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 4 Notable Structural Features of MRP1
(A) Surface representation of the lasso motif. Color schemes: lasso, purple; TMD0, red; TMD1, green; TMD2, blue. (B) Superposition of the lasso motifs of CFTR (cyan) and MRP1 (purple). (C) The “ball-and-socket” joints at the NBD/TMD interfaces. The side chain of F728 is shown as sticks. The s5/h2 elements in NBD2 are highlighted in magenta. In NBD1, the corresponding region is a 4-residue loop, also highlighted in magenta. (D) Superposition of NBD1 onto NBD2 illustrates the absence of s5/h2 in NBD1. The LTC4-bound structure was used to make these figures. See also Figure S3. Cell , e9DOI: ( /j.cell ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 5 Structure of the MRP1-LTC4 Complex
(A) Cartoon representation from within the plane of the membrane with the same color scheme as in Figure 3. LTC4 is shown as sticks, colored by heteroatom (C, yellow; O, red; N, blue; S, gold). (B) EM density of LTC4 in the same orientation as (A). (C) Zoomed in view of the binding site. Viewed from the cytoplasm with interacting side chains shown as sticks. Hydrogen bonds and salt bridges are indicated by dashed lines. Residues that form van der Waals contacts with LTC4 are also indicated. Also shown are S1096, Q1088, and M601, which do not contact LTC4 but can potentially interact with other substrates. (D) Schematic diagram of the LTC4 interactions. The GSH moiety of LTC4 is shown in red and the arachidonic acid moiety in black. Dashed lines represent hydrogen bonds and salt bridges. Residues forming the P-pocket are indicated in blue and the H-pocket residues are in gray. See also Figures S2–S4. Cell , e9DOI: ( /j.cell ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 6 Conformational Changes upon LTC4 Binding
(A) Global superposition of the apo and LTC4-bound structures. (B) Local conformational changes at the substrate-binding site. Arrows indicate movements of the side chains. (C) The two NBDs, viewed from the cytoplasm along the membrane normal. The Cα distances between the Walker A glycine and the serine in the signature motif are indicated. (D) Schematic diagram of the conformational changes. TMD0 is colored red, TM bundle 1/NBD1 is green, TM bundle 2/NBD2 is blue, and LTC4 is shown as yellow sticks. See also Figure S5 and Movies S1 and S2. Cell , e9DOI: ( /j.cell ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure 7 Structural Comparison of MRP1 and P-gp
(A and B) The electrostatic properties of the interior surface of the translocation pathway of P-gp (A) and MRP1 (B), calculated assuming a pH of 7 and a concentration of 0.15 M both (+1) and (−1) ions. Scale: red, negative (−5 kT/e); blue, positive (+5 kT/e). Also shown in (B) are some of the side chains that interact with LTC4. (C and D) Surface representations of the transmembrane regions of P-gp (C) and MRP1 (D). The position of the lipid bilayer is indicated in gray. For these figures, the mouse P-gp structure (PDB 4Q9H) and apo MRP1 were used. Cell , e9DOI: ( /j.cell ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure S1 Data and Model Quality Assessment, Related to Figure 2
(A and B) Plots of the Fourier Shell Correlation (FSC) between reconstructions of two half-datasets. (C and D) FSC calculated between the refined structure and the half map used for refinement (work), the other half map (free), and the full map. Cell , e9DOI: ( /j.cell ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure S2 Density Maps Colored by Local Resolution Estimation Using Blocres, Related to Figures 2, 3, and 5 Cell , e9DOI: ( /j.cell ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure S3 EM Density of Each TM Helix, the Lasso Motif, the NBDs, and the NBD1/TMD Interface of the LTC4-Bound Structure, Related to Figures 2, 3, 4, and 5 Cell , e9DOI: ( /j.cell ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure S4 EM Density of the TMD0 Region, Related to Figures 2, 3, and 5 Cell , e9DOI: ( /j.cell ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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Figure S5 Superposition of the Two Rigid Bodies that Move in Concert upon LTC4 Binding, Related to Figure 6 Cell , e9DOI: ( /j.cell ) Copyright © 2017 Elsevier Inc. Terms and Conditions
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