Insights on Antibiotic Translocation : Molecular Dynamics Simulations

Transcription

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

2 Outline METHODS 1. Model to assess translocation : What can we compare with experiments? 2. Interrelation between Experiments and Simulations Metadynamics : Different reaction co ordinates, free energy quantification Validations for simulation runs ( RMSF, RMSD ) 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 SINGLE MOLECULE EXPERIMENTS : Interruption in ion flow associated with 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,

4 Interrelation between experimental and simulation data Transition Rate Theory : Energy 7 kb T = 1 ns 14 kb T = 1 μ s 21 kb T = 1 ms A G B Reaction Co ordinate K.E.Cooper et al, J. Membrane Biol (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 Z coordinate 1. Free Energy construction in the phase space of the chosen Reaction co ordinates. 2. Each Color Corresponds to 1 Kcal/mol. 9 Kcal /mol. Angle 3. High Intensity of the color correspond to strong interactions.

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

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

10 Area Calculation along OmpF X Ray Structure : 2OMF (PDB ID ) Z axis 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 Hole1. ( Between Z ( 6:6)Š) Useful to calculate the fluctuation of area (with / without) presence of antibiotic with an effort to correlate with BLM experiments. Area (Ų) O.S.Smart et al, Biophysical Journal (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 : Wild Type (WT) 2. D113N_E117Q : Double Mutant (DM) 1.

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 strong minimum at Z = ( 7.8 to 9.8 )Å ang = ( 70 to 90 ) deg Angle The antibiotic makes strong H bonds with residues R42, and switches its H bonded interaction between K80, R168

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 Repulsion effect and low entropy due to unfavorable environment. Distance group down. (min z= 3.8Å) 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 Inventory Interaction map for Minima's near Constriction region : Mini 2 and Mini 3 Mini 2 Mini 3 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 ~2Ų in the constriction region, closure of pore.

19 Comparing WT with DM Mini 1 which lies very much above the constriction region, the WT and DM have nearly similar interaction. ( mutations has not much effect ). 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 experiments (~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 SER125 OH Interaction map for mini's near constriction region

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 ( ) and E117. Orientation perpendicular. Phenyl group slides along the L3 loop Phenyl group top AREA CALCULATIONS area available along Z axis, for the minima of R132a, D113n. Z axis (Å) figure on right represents the sas Area (~ 9 Ų) Area ~ (0 Ų) Area (Ų) for Z= 2 (constriction region) along the simulation time. Area (Ų) figure on right represents the area sliced 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 Area ~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. Moxifloxacin Enrofloxacin Moxifloxacin and Enrofloxacin are zwitterionic and charged zero.

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

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

29 Comparison with fret experiments 1 E (t ) = R 6 (t ) 1+ 6 R0 (t ) Where R06 is the foster radius (E = 0.5 % ) ; and R6 is the distance between the dipole of Trp and Moxifloxacin. E(t) measures the energy transfer between the trp and the antibiotic.

30 Comparison with Fret experiments Moxifloxacin Tryptophane ETrp61=0.957 ETrp214=0.378 High value for Etrp 61 implies difffusion of Further details see poster on Floroquinolones moxi through OmpF

31 Conclusions and Prespectives for Floroquinolones From Simulations : translocation of Moxifloxacin and Enrofloxacin. Thanks to the flouresence property in Floroquinolones, which allow us to compared diretly with the FRET experiments. We observe a high efficiency of energy transfer between Trp 61 and moxifloxacin. Good agreement with experiments. Complete closure of pore and conformational change is the pore (expansion) to accomadate the bulky antibiotic. (agreement with BLM experiments) We extend the methodology from Moxi to Enro and observe similar interactions between the antibiotic and OmpF during the translocation process. (need overlap integral for Enro (from our partners in Porto to calculate the E(t) )

32 4) Cephalosporins CEFPIROME ( CFR) CEFETAMET ( CFT) Charge 0 Charge ( 1)

33 Free Energy Representation CFT WT CFR WT I Z Coordinate V11 ANGLE

34 Preminilary results for CFT Above the constriction region H bond interactions with the stacked arginines, similar conformation for Mini2, Mini3 and Mini4. H contacts with Met 38 Movie : translocation of Cefetamet (CFT)

35 Work In progress CEPHALOSPORINS Cefpirome WT D113A Completed (Eric s Completed (Eric s talk) Cefepime In progress Cefetamet (chagre 1) Preliminary results Ceftizoxime talk) D121A Analysis to completed In progess In progress (translocation) In progress

36 Acknowledgement 1. Thanks to our Network partners : Experimental data 2. Attilio Vargiu : Valuable help Parameterisation of Antibiotics Computing Facility as CASPUR (Rome), CINECA,CYBERSAR (Cagliari ). Financial support