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I Insights on Antibiotic Translocation : Molecular Dynamics Simulations Amit Kumar, Eric Hajjar,Enrico Spiga, Francesa Collu, Paolo Ruggerone & Matteo Ceccarelli Marseille : 11 04-2008 Outline METHODS Model to assess translocation :

  1. I Insights on Antibiotic Translocation : Molecular Dynamics Simulations Amit Kumar, Eric Hajjar,Enrico Spiga, Francesa Collu, Paolo Ruggerone & Matteo Ceccarelli Marseille : 11 –04-2008

  2. Outline METHODS Model to assess translocation : What can we compare with experiments ? 1. Interrelation between Experiments and Simulations 2. Metadynamics : Different reaction co-ordinates , free energy quantification 3. Validations for simulation runs ( RMSF , RMSD ) 4. Results : Successful stories in light with experimental findings : 1 . Carbenicilllin diffusion through WT and D113N_E117Q mutants OmpF 2. Ampicillin with Mutants R132A and D113N 3. Floroquinilones : Comparison between Moxifloxacin and Enrofloxacin 4. Cephalosporins : Comparison between Cefpirome and Cefetamet (preminilary results)

  3. Modeling Translocation From Blockage Interruption in ion flow associated with SINGLE MOLECULE EXPERIMENTS : blockage of antibiotic  BLM experiment : The Rate constant k , can be obtained by noise analysis in frequency spectra . Free energy model : Every 2 blockage corresponds to 1 translocation. Nestorovich et al. PNAS vol . 99 , 15, 9789-9794 2002

  4. Interrelation between experimental and simulation data  Transition Rate Theory : y g r e n ∆G E A B 7 k B T = 1 ns 14 k B T = 1 µs 21 k B T = 1 ms Reaction Co-ordinate K.E.Cooper et al , J. Membrane Biol. 106 95-105 (1988)

  5. Metadynamics Metadynamics : Accelerates the process to be observable within the reach of Standard MD Simulations . Provides Coarse-garined description for the process in phase space defined by a set of reaction ccordinates . Main problem: how to choose the reaction coordinates of a given process ? Laio and Parrinello , 99 , PNAS 2002

  6. Reaction Coordinates Z- coordinate : The axis of diffusion is defined as center of mass (com )of the antibiotic with respect to com of system along z . Angle : Defined as Orientation of molecule's dipole with respect to com of system along z . H-bond : Defined as number of H- bond between the antibiotic and the protein . 0- degree 180- degree

  7. Free Energy Surface Evaluation 1 . Free Energy construction in the phase space of the chosen Reaction co-ordinates . e 2 . Each Color Corresponds to t a n 1 Kcal/mol . i d r o 9 Kcal /mol. 3 . High Intensity of the color o c correspond to strong interactions . - Z Angle

  8. Validation of Simulation runs Root Mean square Fluctuations and Converted B-factors L2 L1 L3 L4 L6 L7 L8 L5

  9. Validation for Simulation Runs (ii) Root Mean Square Deviations

  10. Area Calculation along OmpF X-Ray Structure : 2OMF (PDB ID ) Solvent accessible area was calculated  using in house built program :Hope for the X-Ray structure of OmpF .  Good agreement with the well known program Hole 1 . ( Between Z (-6:6) Å ) Z - a x i Useful to calculate the fluctuation of  s area (with / without) presence of antibiotic with an effort to correlate with BLM experiments. Area (Ų) O.S.Smart et al , Biophysical Journal 65 2455-2460 (1993)

  11. I) The case of Carbenicillin Carbenicillin (CRB) has charge of -2 . The diffusion of CRB through ompf is difficult due to the repulsion between the carboxy group and the residues 113 and 117 near the constriction region. On Screening the repulsion : By Mutation of the residues D113  N113 and E117  Q117 can we expect the translocation process for CRB ? To answer our question : OmpF Investigated : 1. Wild Type (WT) 2. D113N_E117Q : Double Mutant (DM)

  12. Free Energy Surface Evaluation Angle  Carbenicillin with Wild Type (WT) mutant no translocation .  Carbenicillin with D113N_E117Q mutant translocation

  13. Movies For WT and D113N_E117Q OmpF

  14. Why No translocation through WT OmpF Mini -1 Z-axis The antibiotic makes strong H-bonds The strong minimum at with residues R42 , and switches its H- Z = ( 7.8 to 9.8 ) Å bonded interaction between K80 , R168 ang = ( 70 to 90 ) deg Angle

  15. Observation along the trajectory for WT 1. For most part of the total trajectory  ( ~20ns) the antibiotic stays in Mini-1. Favorable Interaction of oxygens with  K80 , 167, 168 . Move towards constriction region : Snapshot for a configuration where CRB arrives  near constriction region parallel to axis of diffusion , phenyl C group down. (min z= 3.8 Å ) Repulsion effect and low entropy due to  Distance unfavorable environment. Figure C on the right depicts distance between D113 – COO (black) and E117 – COO (red) Time (20ns)

  16. Translocation for CRB with DM Z-axis Angle

  17. Observation along trajectory of translocation I nventory Interaction map for Minima's near Constriction region : Mini 2 and Mini 3 Mini -3 Mini -2  In the two minima's the oxygens of the antibiotic makes H-bonded interactions with the stacked arginines ( Switching between R132 , R82 , R42 , K16 ) and in particular we observe the oxygens making interactions with N113 and Q117 .

  18. Area Calculation for the Minima's for DM Z-axis (Å) Area (Ų) The Mini-2 and Mini-3 which lie near/on the constriction region in presence of antibiotic the area is as low as ~2 Ų in the constriction region , closure of pore.

  19. Comparing WT with DM  Mini-1 which lies very much above the constriction r egion , the WT and DM have nearly similar interaction . ( mutations has not much effe ct ) .  For WT CRB does not arrive near the constriction region , due to repulsion and supported low entropy for CRB created by the residues D113 , E117 .  For DM near constriction region : No repulsion effect + Higher Entropy = Translocation .  Interactions with N113 and Q117 essential for translocation .

  20. Comparing with experiments for CRB  BLM experiments : our partners ( Bremen ) 1. WT ---------> No Blockage -------> No Translocation. 2. DM ----------> No Blockage --------> No Translocation.  Simulation Results : 1. WT --------------> No Translocation 2. DM ---------> Translocation.  Area calculation : in presence of antibiotic area ~2Ų , the pore is blocked perhaps for a timescale not resolved by exper iments (~10µs) .

  21. The effects of Ompf mutants on ampicillin diffusion BASIC RESIDUE MUTATION : R132A change in  charge + size ACIDIC RESIDUE MUTATION : D113N change in charge View of Constriction Zone

  22. Free Energy Surface Z-coordinate Mini Mini Angle

  23. Interaction map for mini's near constriction region SER125-OH

  24. Observation along trajectory of translocation For Mutation of R132A –movement of ampicillin towards A132 – finds more space  H- bonded interaction with residues localized in neighbour-hood of A132 . The orientation is parallel to axis of diffusion. ( phenyl group down) On Mutation D113N – H-bonded interactions with stacked arginines (42 82 132 )  and E117 . Orientation --- perpendicular . Phenyl group slides along the L3 loop ---- Phenyl group top AREA CALCULATIONS Z-axis (Å) figure on right represents the sas Area (~ 9 Ų) Area ~ (0 Ų) area available along Z- axis , for the minima of R132a , D113n . Area (Ų) figure on right represents the area sliced Area (Ų) for Z= -2 (constriction region) along the simulation time . Simulation time

  25. Comparison with Experiments  BLM experiments (Bremen) 1. D113N ------- Slight Increase in background noise . 2. R132A -------No Blockage ----- No translocation  Simulation Results 1. D113N ----- Translocation ~ Area ~ 0Ų 2. R132A ---- Translocation A rea ~9Ų  Liposome swelling assay experiments (Bremen and Porto) : Swelling rate for mutants R132A and D113N more compared to wild type , confirms translocation of ampicillin for the mutants .

  26. 3) Flouroquinolones Floroquinolones are more bulky and hydrophobic compared to the family of penicillin’s. Enrofloxacin Moxifloxacin Moxifloxacin and Enrofloxacin are zwitterionic and charged zero .

  27. Free Energy Represenatation MOXIFLOXACIN ENROFLOXACIN 3 Translocation with coo - group down . Translocation with coo - group up.

  28. Comparison with BLM experiments Area calculation for the constriction region all along the trajectory of translocation . M oxifloxacin Enrofloxacin Complete closure and and conformational change conformational change Complete closure of the pore during the translocation of the pore during the translocation

  29. Comparison with fret experiments 1 Where R 0 6 is the foster radius (E = 0.5 % ) ; and R 6 is the = ( ) E t distance between the dipole of Trp and Moxifloxacin. ( ) 6 R t + 1 E(t) – measures the energy transfer between the trp and the ( ) 6 R t antibiotic . 0

  30. Comparison with Fret experiments Moxifloxacin Tryptophane E Trp61 =0.957 E Trp61 =0.957 E Trp214 =0.378 E Trp214 =0.378 High value for E trp 61 implies difffusion of moxi through OmpF Further details see poster on Floroquinolones

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