Characterizing Structural Transitions of Membrane Transport Proteins at Atomic Detail Mahmoud Moradi

Transcription

1 Characterizing Structural Transitions of Membrane Transport Proteins at Atomic Detail Mahmoud Moradi NCSA Blue Waters Symposium for Petascale Science and Beyond Sunriver, Oregon May 11, 2015

2 Outline Introduction GlpT transporter Transport cycle thermodynamics Methodology Empirical search for reaction coordinates using nonequilibrium simulations Iterative path-finding algorithms and free energy calculations Reconstructed thermodynamic cycle of GlpT Free energy profile along the cycle Global and local conformational changes and their coupling

3 Membrane Transporters Glt GlpT MsbA Transporters: Membrane proteins which actively and selectively transport materials (proton, ions, small molecules) across cell membranes. Active transport: Pumping substrates against their concentration gradient (from low to high concentration). Source of energy: metabolic energy, e.g. from ATP hydrolysis (primary). electrochemical gradient of an ion (secondary).

4 Alternating-Access Mechanism Membrane transporters rely on large-scale conformational changes to alternate between inward-facing (IF) and outward-facing (OF) states to pump the substrate against its concentration gradient, without being open (having the binding site accessible) to both sides of the membrane simultaneously. out in out in (OF a ) (IF a ) OF apo OF bound IF apo IF bound (OF b ) (IF b ) out in out in

5 Glycerol-3-phophate (G3P) transporter (GlpT) Major facilitator superfamily (MFS) Secondary active transporter Crystalized in the IF state. periplasm cytoplasm P i G3P GlpT transports G3P using P i gradient. Huang, et al., Science 301, 616 (2003). P i :P i exchanger (in the absence of organic phosphate) Rate-limiting step: IF-OF interconversion.

6 Transport cycle thermodynamics H7 H1 P i OF a IF a H11 H5 OF b IF b

7 Free Energy Transport cycle thermodynamics a: apo b: bound Free Energy barrier Transition State OF b IF b OF a IF a Reaction Coordinate Lemieux, et al., Curr. Opin. Struct. Biol. 14, 405 (2004). Law, et al., Biochemistry 46, (2007).

8 Full thermodynamic cycle H7 H1 P i OF a IF a H11 H5 OF b IF b

9 H7 the only available crystal structure H1 P i OF a IF a H11 H5 OF b IF b

10 Key Challenge: Slow dynamics Timescale gap between feasible all-atom molecular dynamics (MD) simulations and actual functionally relevant biomolecular processes. Sampling Strategies: Long simulation application-specific computers Multiple-copy simulations distributed computing Enhanced sampling biased/adaptive simulations Loosely-coupled multiple-copy algorithms (petascale computing)

11 H7 Step 1: OF a IF a H1 P i OF a IF a H11 H5 OF b IF b

12 Empirical search for reaction coordinates and biasing protocols Work Optimized Protocol Path-Refining Algorithms Free Energy Calculations Theory/Method: Moradi et al., CPL (2011) Moradi et al., JCP ,5 (2014) Moradi et al., JCTC (2014) Reaction Coordinate Application: Moradi et al., PNAS (2009) Moradi et al., NAR (2013) Moradi et al., PNAS (2013)

13 Empirical search for reaction coordinates and biasing protocols: IF OF Nonequilibrium Work about 100 simulations with different protocols

14 Path-finding algorithms String Method (finding approximate minimum free energy pathways on high-dimensional spaces) A pathway is represented by a string, i.e., an ordered series of images product regions. connecting reactant and The string is iteratively updated according to some ``rule until converges to a stationary solution: Maragliano, Fischer, Vanden-Eijnden, and Ciccotti J. Chem. Phys. 2006, 125, Ren, Vanden-Eijnden, Maragakis, and E J. Chem. Phys. 2005, 123, Vanden-Eijnden and Venturoli; J. Chem. Phys. 2009, 130,

15 Path-finding algorithms String Method with Swarms of Trajectories (SMwST): For each image tens of copies are launched: Start with an initial string (1) Restrain M copies of each image at the current (2) Release the restraint (3) Update the centers: (4) Reparametrize Pan, Sezer, and Roux J. Phys. Chem. B 2008, 112, Collective variables: {Q} = {Q 1,Q 2,...,Q 12 } orientation quaternions of all helices Number of replicas: 50 X 20 = 1000 Simulation time: 1 ns/replica

16 Free Energy Free energy calculations Bias-exchange umbrella sampling (BEUS) (Loosely coupled multiple-copy MD) Umbrella sampling U B (x t i ) = 1 k(x t 2 i -x i ) 2 Replica-exchange MD Reaction coordinate Replica2 Replica1

17 Iterative path-refining algorithms and free energy calculations BEUS Bias-Exchange Umbrella Sampling (Free Energy Calculation) MCA SMwST PHSM Post-Hoc String Method (Path-Finding Algorithm) String Method with Swarms of Trajectories (Path-Finding Algorithm) Analysis Technique MCA

18 H7 Step 1: OF a IF a H1 P i OF a IF a H11 H5 OF b IF b

19 H7 Step 2: IF a IF b H1 P i OF a IF a H11 H5 OF b IF b

20 H7 Step 3: OF a OF b H1 P i OF a IF a H11 H5 OF b IF b

21 H7 Step 4: OF b IF b H1 P i OF a IF a H11 H5 OF b IF b

22 Transition Technique Collective Variables # of Replicas Runtime 1 BEUS (Q 1,Q 7 ) ns = 0.5 ms 2 IF a OF a SMwST {Q} ns = 1 ms 3 BEUS {Q} ns = 1 ms 4 BEUS Z IF a IF Pi ns = 1.2 ms 5 b BEUS ({Q}, Z Pi ) ns = 1.2 ms 6 BEUS Z OF a OF Pi ns = 1.2 ms 7 b BEUS ({Q}, Z Pi ) ns = 1.2 ms 8 BEUS (Q 1,Q 7 ) ns = 0.5 ms 9 BEUS Z Pi ns = 0.5 ms IF b OF b Simulation protocols 10 2D BEUS ( RMSD, Z Pi ) ns = 1 ms 11 SMwST ({Q}, Z Pi ) ns = 1 ms 12 BEUS ({Q}, Z Pi ) ns = 1 ms Each replica consists of ~150,000 atoms 13 Full Cycle BEUS ({Q}, Z Pi ) ns = 7.5 ms GlpT Crystal Structure BEUS SMwST Total Simulation Time Full Cycle PHSM 18.7 ms Nonequilibrium

23 H7 H1 P i OF a IF a H11 H5 OF b IF b

24 OF a IF a OF b IF b

25 Free Energy (kcal/mol) OF a binding unbinding Image Index TS a TS b OF b Image Index IF b IF a unbinding binding

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29 Distinct conformational transition pathways Quaternion-based principal components (QPCs) represent different modes of concerted motions of transmembrane helices. with substrate without substrate

30 Characterizing protein local conformational changes within the lumen: E299 R269 D274 K46 Salt bridges stabilizing different conformations. Residues involved in binding. R269 OF a IF a P i K46 R45 R45 Conformational dynamics of the binding site OF b IF b P i K80

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32 Summary Reconstructed thermodynamic cycle of GlpT Alternating access mechanism characterized (atomic level) Substrate binding lowers the IF-OF transition barrier Substrate binding changes the IF-OF transition pathway Coupling between local and global conformational changes Reconstructing transport cycles in membrane transporters using enhanced sampling techniques and petascale computing Moradi M., Enkavi G., and Tajkhorshid E., under review by Nature Communication (2015).

33 Acknowledgement Emad Tajkhorshid Giray Enkavi Tajkhorshid Lab, Beckman Institute, UIUC Sundar Thangapandian, Jing Li, Po-Chao Wen, Josh Vermaas, Noah Trebesch, Javier Baylon, Mrinal Shekhar, Steven Wang

34 Rocker-switch model Rocker-Switch Model