Equilibrated atomic models of outward-facing P-glycoprotein and effect of ATP binding on structural dynamics (Supplementary Information)

Transcription

1 Equilibrated atomic models of outward-facing P-glycoprotein and effect of ATP binding on structural dynamics (Supplementary Information) Lurong Pan 1 and Stephen G. Aller 2 * 1,2 Department of Pharmacology and Toxicology, University of Alabama at Birmingham, th Street South, Birmingham, AL, USA *Correspondence to sgaller@uab.edu Text S1. Equilibration and sampling size The protein C α root-mean-square deviation (RMSD) revealed that the major conformational change occurred in the first 100 ns of simulation and the protein reached different equilibrium states after 100 ns (Figure S1). The equilibrated RMSDs of minor changes within a 1 Å range indicated that the protein adopted certain predominant meta-stable conformations and oscillated between them. The equilibration findings showed that the time scale of the simulations was sufficiently long to reveal various meta-stable conformations that were independent from the starting structure. Both apo-pgp and MgATP-Pgp were simulated in triplicate to acquire sufficient sampling and to avoid biased conclusions. According to the Ergodic hypothesis, the time average is equivalent to the ensemble average. Therefore, the average of the trajectories from triplicate simulations over time was used in order to calculate the dynamic features of the each model in an ensemble with considerable sampling size. Representative microstates with distinct conformational features were also analyzed. Text S2. Molecular dynamic simulations Atomic coordinates of outward-facing Pgp with/without MgATP 2- (apo-pgp/mgatp-pgp) was first placed in a POPC bilayer. The dimension of the inward-facing crystal structures was (Å) and the dimension of the outward-facing homology structure was (Å). The change of dimension involves the fluctuation of TMDs from inward-facing to outward-facing conformations and potential rotation of the protein around the bilayer center normal (z-axis). Therefore, the lipid bilayer was built as (Å) in order to accommodate all possible dynamic conformations. POPC was used as the sole bilayer-forming lipid in our simulations. The long principal axis of the protein was aligned with the bilayer center normal (z-axis). Previous studies indicated that Trp residues are positioned to form hydrogen bonds between the -NH group on the indole ring and carbonyl group of the glyceride on the lipid while the majority of the indole rings are immersed towards the hydrophobic side of the bilayer 1. In order to achieve a proper lipidprotein interface for both leaflets of the lipid bilayer, residues Trp208, Trp311 and Trp851 were aligned with the glyceride groups of the outer leaflet of membrane whereas residues Trp44, Trp132, Trp228, Trp694 and Trp704 were aligned with the glyceride groups of the inner leaflet of the membrane. After the protein was positioned in the lipid bilayer correctly, the overlapping lipid groups were removed with 0.6 Å cutoff distance from the protein. The membrane-embedded protein was then dissolved in a water box without removing the water molecules inside the bilayer. Eliminating water within the lipid would introduce micro vacuum pockets in the initial structure, and we therefore fully solvated the lipid to allow unbiased adjustment of the system to a proper lipid solvation state. After testing different system sizes, we were able to determine that larger padding

2 distances do not provide any more stability or any apparent advantage over standard padding, allowing us to proceed with a smaller overall simulation box for our planned experiments. In this smaller box, the dimensions adjusted to ~ (Å) after equilibration. The padding distance maintains >12 Å from every direction in equilibrium. Free ions with the concentration of 0.15 M NaCl and M of MgCl 2 were added to mimic physiological ionic condition and to neutralize the total system charge. The total number of atoms in the system is ~209K for each model. Conventional MD simulations were performed for all models to examine the thermodynamic stability and potential structural transformation for each starting conformation. The simulations were performed at isothermal isobaric ensemble (NPT ensemble) at body temperature of 310 and 1 atmosphere pressure. The constant temperature was maintained with Langevin dynamics using a damping coefficient of =1 ps -1. Constant pressure ( bar) was maintained using Nosé- Hoover Langevin piston pressure control 2,3 with the Langevin piston period of 200 fs and langevin Piston decay of 100 fs. The time steps were set to 2 fs for the entire simulation and trajectories at every 5000 steps (10 ps) were saved for analysis. Each system was first subjected to conjugate gradient minimization for steps (20 ps). Then, various groups of harmonic constraints were applied for a multi-step simulation to achieve final equilibrium. The system temperature was increased from 0 to 310K at a rate of 1K/ps with the constraints (force constant k=1kcal/mol/ Å 2 ) on protein, ligands and the P atoms of lipids. We then maintained the same constraints for another 5 ns simulation to achieve the equilibrium of water. The constraints on P atoms of lipids were then removed and simulated for another 5 ns to equilibrate the lipid species. The last step was to keep starting constraint (force constant k=5 kcal/mol/å 2 ) on the protein backbone and ligands only, gradually removing the constraints at a speed of 0.5 kcal/mol/å 2 per 500 ps. This step accomplished the maximum packing between the lipid and the protein. In order to observe potential conformation change under conventional unbiased MD simulations, a 200 ns production phase was performed without any constraint on each model. Due to the unconstrained nature of conventional simulations, nucleotide-bound and nucleotide-free Pgp simulations were each performed in triplicate to avoid conclusions based on spurious events. All atomistic MD simulations were performed using NAMD2.9. All MD simulations utilized CHARMM22 with the CMAP correction of all-atom protein force field 4 and CHARMM36 all-atom lipid force field 5. The combination of CHARMM22 with CMAP correction for protein and CHARMM36 for lipid performed well for a similar recent transmembrane protein simulation analysis 6. The force field of ATP 4- was implemented by CHARMM36 7. For both electrostatic and van der Waals calculations, 8 Å switching distance and a 12 Å cutoff distance were used. The particle mesh Ewald (PME) method 8 was employed to calculate the long-range electrostatic interactions with 1.0 grid spacing. The TIP3P explicit water model 9 was utilized. Secondary structural analyses was based on STRIDE algorithm 10. Electrostatic potential calculation used APBS 11 plugin within PyMOL. PyMOL and VMD were used for modeling and molecular image creation.

3 Figure S1. Root-mean-square deviation (RMSD) of protein C α over time. The protein C α RMSDs reveals that the major conformational change took place in the first 100ns of simulation while the protein reach equilibrium states after 100ns. The equilibrate RMSDs with minor changes within 1 Å range indicated that the protein adopt certain predominant meta-stable conformations and oscillate between multiple meta-stable states.

4 Figure S2. Secondary structure component of the apo-pgp run1 (A), run2 (C), run3(e) and MgATP-Pgp run1 (B), run2 (D), run3 (F) over time. Secondary structures were calculated using the STRIDE algorithm. The secondary structure component (alpha helix in blue, turn in red, coil in green, beta sheet in purple, 3_10 helix in orange and beta bridge in light blue) remains consistent during the entire simulation.

5 Figure S3. Secondary structural components of the homology model, X-ray Crystallography structures and equilibrate trajectories from MD simulations. Secondary structures were calculated using the STRIDE algorithm. Homology in blue is the outward-facing mouse-pgp homology model built in this study. Sav1866 (PDB: 2ONJ) in red is the X-ray crystal structure of the outward-facing conformation of Sav1866, mouse-pgp (PDB: 4M1M-A) in green and mouse- Pgp (PDB:4M1M-B) in purple are inward-facing conformations of mouse-pgp from two slightly different X-ray crystal structures. apo-pgp run1-3 in aqua blue, orange and light blue and MgATP- Pgp run1-3 in pink, light green and light purple are conformations from MD simulations. The conformations from MD simulation use average from the last 50 ns (150ns -200ns). The secondary structures of Pgp are conserved (<5% difference) 1) across species between mouse-pgp and Sav1866 and 2) are conserved between conformations: outward-facing conformations (homology model, Sav1866, PDB:2ONJ), intermediate conformations from MD simulations (apo-pgp run1-3, MgATP-Pgp run1-3) and the inward-facing conformations (mouse-pgp PDB: 4M1M-A,B).

6 Figure S4. Per-residue secondary structure probability over time (dynamic secondary structure). Secondary structures were calculated using the STRIDE algorithm. The measurement of dyanmic secondary structure of each residue are based on the last 50 ns equilibrated trajectories averaged over the triplicate simulations for both apo-pgp (A, B) and MgATP-Pgp models (C, D). Panels A and C: first "half" of mouse Pgp. Panels B and D: second "half" of mouse Pgp. Alpha helix in blue, turn in red, coil in green, beta sheet in purple, 3_10 helix in orange and beta bridge in light blue. The figure shows the percentage of time for each residue adopting a certain secondary structure.

7 Figure S5. ATP-binding pocket specifics. The MgATP2- is drawn in licorice with H in white, N in blue, C in cyan, O in red, P in gold and Mg2+ in pink. The protein is shown in cartoon representation with half1 in pink and half2 in ice blue. (A) shows the overview of an MgATP2- complex binding to the mouse P-pg at site 2. (B) shows and H-bond of ATP(Adenine-NH2)-D160/D801(OE). (C) shows multiple H-bonds between the LSGGQ motif with ATP including ATP(H2 )-Q531/Q1176 (OE), ATP(O3 )-Q531/Q1176 (HE), ATP(Oγ)-S528/S1173(HG), ATP(Oγ)-G529/G1174(HN), ATP(Oγ)-G530/G1175(HN). (D) shows five coordination bonds between Mg2+ and the following atoms: E552/E1197 (OE1, OE2), S430/S1073 (OG), and ATP (Oβ, Oγ). (E) shows the H-bond network between MgATP2- and Walker A motif including ATP(Oγ)-S425/S1068(HG), ATP(Oγ)K429/K1072(HZ), ATP(Oβ)-K429/K1072(HZ), ATP(Oβ)-S430/S1073(HN), ATP(Oα)T431/T1074(HG). (F) shows the aromatic interactions between ATP (adenine ring)-y397/y1040 (ring). The H-bonds and coordination bonds are in the range between Å in the initial structure.

8 Figure S6. Electrostatic potential of two halves of mouse Pgp and Sav1866. The crystal structure of Mouse Pgp (PDB: 4M1M) and crystal structure of Sav1866 (PDB: 2ONJ) are drawn in surface using Van der Waals radius. Vacuum electrostatics is calculated using the APBS plugin in PyMOL. Blue/Red color gradient is used to show the charge distribution from the positive end in blue to the negative end in red. The comparison between core regions of TMDs in the Mouse P-pg (A1, A2) and Sav1866 (A) shows an overall neutral region in Mouse P-pg but highly charged region in Sav1866. On the other hand, the interface between two NBDs in mouse P-pg (B1, B2) are more charged than that of Sav1866 (B).

9 Figure S7. Sequence alignment and homology structure prediction. (A) Sequence alignment of a mouse Pgp (first lines) monomer and a Sav1866 (second lines) dimer containing minimal gaps. The linker region (residues ) of mouse Pgp was removed and two tandem copies of Sav1866 were generated. The two resulting amino acid sequences were aligned using the EMBOSS Needle pairwise alignment tool at the EMBL ( using PAM500 similarity matrix, a gap opening penalty of 50 and a gap extension penalty of 10. The resulting alignment had a good amino acid sequence identity of 30%, a similarity of 63%, and only contained 8 gaps (ovals). Due to poor structural conservation of the first and seventh transmembrane helix (TM1, TM7) upon inspection of the inward-facing conformation (PDB: 4M1M), TM1, TM7 and the first extracellular loop (ECL1) of Pgp (residues 1-106) and ECL4 (residue 692 to 747) were removed (top sequence in rectangles). Likewise, Sav1866 TM1 and ECL1 (residues 1-59) were also removed (bottom sequence in rectangles). Four C-terminal residues of Pgp ( ) were removed. (B) The homology model (in orange cartoon) of mouse-pgp has 0.78 Å α-carbon RMSD from the template structure of Sav1866 (in cyan cartoon).

10 Figure S8. Structural alignment and final homology structure. Missing fragments were truncated in the homology model from previous step (Figure S7)(in silver cartoon) using mouse-pgp crystal structure (PDB: 4M1M) (in magenta cartoon) to correctly insert the gap residues. Structure fragments of residue 30 to 107 (A) and residue 681 to 747 (B) were aligned onto the homology structure without disturbing the integrity of the connecting helices. Fragment 368 to 385 was replaced to model missing residue Phe374 (C), Fragment 1009 to 1032 was replaced to model missing residue 1017 (D). Fragment 390 to 410 was replaced to model missing residue Pro398 (E). Fragment 1034 to 1054 was replaced to model residue Pro1044 (F). Fragment 793 to 808 was replaced to model missing residues Asp801 and Asp802 (G). Fragment 1129 to 1139 was replaced to model missing residue 1137, 1138(H).

11 Figure S9. Philosophy of the homology modeling of mouse-pgp outward-facing conformation. The homology model of the outward-facing conformation (C) of mouse Pgp was generated based on both sequence alignment with Sav1866 outward-facing conformation (A) crystal structure (PDB: 2ONJ) in silver and regional structural alignment with inward-facing conformation (B) of the same mouse-pgp (PDB: 4M1M) in magenta.

12 Figure S10. Problematic homology model generated from MODELLER. (I) Problematic homology model generated from MODELLER using the sequence alignment in Figure S2: fragments with problems are labeled in red. (A): incorrect EHL1 sequence residue , the correct loop sequence is (residue 86-92), the fact mouse Pgp has longer TM1 and TM2 than Sav1866. (B) Incorrect EHL3, sequence residue , the correct loop sequence is residue (C) Incorrect orientation after insertion of gap residue Pro398, the loop should point towards the ATP binding pocket. (D) Incorrect orientation after insertion of gap residue Pro1044, the loop should point towards the ATP binding pocket. (E) Poor helix loop orientation after insertion of gap residue Ser1137 Tyr1138. (F) Original helix broken by the poor modeling of gap at Asp801, Asp802. (G)(H) Poor loop orientation after insertion of Phe 374 and Tyr 1017.

13 Figure S11. Morph calculation between inward-facing conformation (A) (PDB: 4M1M) and outward-facing conformation homology model (E). (B), (C), (D) are morph intermediate structures between the two conformations. Secondary structure remained during the conformational change. Secondary structures are calculated using STRIDE algorithm: alpha helix in purple, 3_10 helix in blue, turn in white, coil in cyan, beta sheet in yellow, beta bridge in tan.

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