Refined structures of mouse P-glycoprotein

Abstract

The recently determined C. elegans P-glycoprotein (Pgp) structure revealed significant deviations compared to the original mouse Pgp structure, which suggested possible misinterpretations in the latter model. To address this concern, we generated an experimental electron density map from single-wavelength anomalous dispersion phasing of an original mouse Pgp dataset to 3.8 Å resolution. The map exhibited significantly more detail compared to the original MAD map and revealed several regions of the structure that required de novo model building. The improved drug-free structure was refined to 3.8 Å resolution with a 9.4 and 8.1% decrease in R(work) and R(free), respectively, (R(work) = 21.2%, R(free) = 26.6%) and a significant improvement in protein geometry. The improved mouse Pgp model contains ∼95% of residues in the favorable Ramachandran region compared to only 57% for the original model. The registry of six transmembrane helices was corrected, revealing amino acid residues involved in drug binding that were previously unrecognized. Registry shifts (rotations and translations) for three transmembrane (TM)4 and TM5 and the addition of three N-terminal residues were necessary, and were validated with new mercury labeling and anomalous Fourier density. The corrected position of TM4, which forms the frame of a portal for drug entry, had backbone atoms shifted >6 Å from their original positions. The drug translocation pathway of mouse Pgp is 96% identical to human Pgp and is enriched in aromatic residues that likely play a collective role in allowing a high degree of polyspecific substrate recognition.

Keywords: P-glycoprotein; SAD phasing; X-ray crystallography; multidrug resistance; protein modeling.

Figure 1

Comparison of experimental electron density maps. Left panels represent the highest resolution map published in Aller et al. produced from multiwavelength anomalous dispersion (MAD) phasing using “Crystal 2.” Right panels represent the new map produced from single-wavelength anomalous (SAD) phasing. The new map was produced by phasing 12 original mercury sites and the λ₁ dataset using phenix.phaser. The final model-free phases were obtained using Wang density modification and non-crystallographic symmetry operators as described in materials and methods. Bottom panels include the original model (left, 3G5U) and the improved structure (right, 4M1M) in gray sticks. The four panels are shown in wall-eyed (divergent) stereo view. Each map is contoured at 1σ.

Figure 2

Improved structure of mouse P-glycoprotein. Wall eyed stereo view of one molecule of the asymmetric unit. The improved structure is displayed and is colored by the distance each C_α has moved from the original position according to the color bar at the top of the figure. Red, orange, and yellow colors represent C_α positions that have changed the most upon correction (up to 8 Å). The changes to the improved mouse Pgp structure are global in scope, with the greatest change to the position of transmembrane helix 4 (TM4). All twelve transmembrane domains (TM1-TM12), and the two nucleotide binding domains are labeled (NBD1, NBD2).

Figure 3

Validation of N-terminus and TM Registry Shifts. Using a Cysless mouse Pgp as template, three additional mutants were constructed, crystallized, and labeled with mercury. (A) A32C mutation near the N-terminus; (B–D) A216C/A244C double mutation; (E–G) A280C/I302C double mutation. The original Pgp structure (PDB 3G5U) is shown in brown ribbons and the improved Pgp structure (PDB 4M1M) is shown in green. Anomalous difference density for all mutants is shown in magenta mesh. (A) view of the N-terminus looking toward the “elbow” helix. Anomalous difference density is contoured at 6 σ. (B) View of entire TM4 for the A216C/A244C double mutant. (C) Zoom view of Ala-216 shown in spheres and labeled for original and improved structures. (D) Zoom view of Ala-244. Anomalous density in panels B–D is contoured at 4 σ. (E) View of entire TM5 for the A280C/I302C double mutant. (F) Zoom view of Ala-280. Panel (H): Zoom view of Ile-302. Anomalous density in panels E-G is countered at 6 σ.

Figure 4

Agreement of Improved Mouse Pgp Structure with Biochemical Data. (A) overall structure of mouse Pgp. (B and C) pairs of residues in TMDs that formed disulfide bonds (green line) when mutated to cysteines., (D and E) pairs of residues at NBD-IH interfaces that were crosslinked., (F) Wall-eyed stereo view of the drug transport pathway. Mouse Pgp residues corresponding to drug interacting residues from human Pgp biochemical studies– are labeled and shown as magenta balls. The non-protected residues Tyr 114, Val 121, Val 129, Cys 133, Gln 191, Ile 293, Gly 296, Ala 297, Leu 300, Ala 304, Ala 307, Phe 310, Ser 725, Phe 755, Ser 762, Gly 770, Leu 829, Phe 833, Ile 836, Ala 837, Gly 840, Thr 841, Ile 843, Ile 844, Ile 845, Ala 867, Ser 939 and Phe 953, are shown in gray.,

Figure 5

Amino acid residues involved in the drug translocation pathway for mouse P-glycoprotein. Residues were selected for the Venn diagram if they are 5 Å or less from the cyclic peptides, QZ59-RRR and QZ59-SSS, in the improved mouse Pgp structures or residues involved in drug interactions as determined by previous biochemical studies. Only two amino acid residues in the entire drug translocation pathway are nonidentical between mouse- and human Pgp (human Pgp residues and numbering shown in parentheses). ^aCys mutant had reduced ATPase when exposed to MTS-verapamil; ^bInteraction also observed with vinblastine and colchicine when mutated to cysteine; ^cInteraction also observed with rhodamine when mutated to cysteine; ^dCys mutant had permanent ATPase in MTS-verapamil.

Figure 6

Interdomain polar contacts in the improved mouse P-glycoprotein structure. (A and B) View of IH1-IH4-NBD1 polar contacts in molecules “A” and “B” of the asymmetric unit, respectively. (C and D) View of IH2-IH3-NBD2 polar contacts in molecule “A” and “B,” respectively. (E) Incorrect modeling of the TM4-TM5 region connecting IH2 in the original mouse Pgp structure. The Glu 252–Arg 272 pair, just above IH1, was modeled more than 11 Å apart in the original structure. (F) A salt bridge between Glu 252 and Arg 272, required for proper folding of Pgp, is shown for one molecule of the asymmetric unit in the improved structure.

Figure 7

Mouse Pgp NBD1-IH4 Interface. Experimental electron density from SAD phasing is shown in blue mesh. The interface is formed by key interactions with Tyr 486 on NBD1 and surrounding residues from extensions of TM 10, 11, and IH4. The interaction energy of the folded interface is largely derived from Van der Waals contact, π–π interactions, and polar contacts. The interface is reasonably well conserved between mouse Pgp (mPgp) and human CFTR (mPgp Phe 476 corresponds to hCFTR Met 498, mPgp Tyr 486 corresponds to hCFTR Phe 508, mPgp Thr 902 corresponds to hCFTR Thr 1064, mPgp Val 903 corresponds to hCFTR Leu 1065, mPgp Arg 908 corresponds to hCFTR Arg 1070, mPgp Phe 912 corresponds to hCFTR Phe 1074, and mPgp Met 915 corresponds to hCFTR Leu 1077). The figure is shown in wall-eye stereo.