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Replica Exchange Simulations of Protein-Protein Binding and Multi-protein Complex Formation

Replica Exchange Simulations of Protein-Protein Binding and Multi-protein Complex Formation. Youngchan Kim and Gerhard Hummer Laboratory of Chemical Physics National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health. February 3, 2009

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Replica Exchange Simulations of Protein-Protein Binding and Multi-protein Complex Formation

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  1. Replica Exchange Simulations of Protein-Protein Binding and Multi-protein Complex Formation Youngchan Kim and Gerhard Hummer Laboratory of Chemical Physics National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health February 3, 2009 NIH Biowulf Symposium

  2. Background • Many biological functions are carried out by large, multi-protein assemblies • DNA transcriptional regulation • Signal transduction • Nuclear pore complex • Membrane-protein trafficking • Viral entry and release ESCRT machinery (Membrane trafficking)

  3. Motivation Understanding structure and dynamics of multi-protein assemblies • Many multi-protein assemblies form only transiently • Held together by relatively weak pairwise interactions (Kd > 1mM) • Multi-protein assemblies contain unstructured regions • Flexible polymeric linkers connecting structured domains • Challenges for traditional structural approaches • X-ray crystallography: Difficult to crystallize weak complexes with unstructured regions • NMR spectroscopy: Size limits • Electron microscopy: Trapping of functional assemblies • New opportunities for modeling, simulation, and theory! • Complement experiments • Provide predictions, insights, and new directions

  4. Outline • Model and Method: • Validation: structures and binding affinities • Structure and dynamics of multi-protein assemblies • Vps27/Hse1: ESCRT protein sorting machinery • Collaboration with James H. Hurley, NIDDK • Transient encounter complexes in protein-protein complex formation • Paramagnetic relaxation enhancement NMR of protein-protein complexes • Collaboration with G. Marius Clore, NIDDK

  5. Ca Tested for protein folding membrane Coarse-grained model for multi-protein assemblies • Residue-level (Ca only) coarse-graining • Rigid body for structured domains • Transferable energy function • Long-range Debye-Hückel electrostatic interactions • Residue-dependent short-range interactions (Miyazawa-Jernigan statistical contact potentials) • Experimental inputs: Lysozyme osmotic protein second-virial coefficient and Ub-CUE proteinbinding affinity • Flexible linkers: polymer model • Harmonic stretching potential • Bending potential • Torsion angle potential • Membrane interactions • Planar membrane • Short-range interactions between residues and membrane • Electrostatic interactions (YCK, Hummer, J. Mol. Biol. 375, 1416, 2008)

  6. Simulation method • Replica exchange Monte Carlo • Twenty replicas at different temperatures • Enhances equilibrium sampling • Implemented in the parallel architecture of Biowulf cluster (YCK, Hummer, J. Mol. Biol. 375, 1416, 2008)

  7. Native-like Native-like Native-like DRMS 1 å = - exp model d d ij ij N i , j Energy Validation: complex structure Ub/CUE Ub/UIM1 Ub/UBA Ub/GAT Ub/DUIM Ub/UIM2 UbL/UBA UbL/UIM UbL/UIM2 Distance to native structure (YCK, Hummer; J. Mol. Biol. 375, 1416, 2008)

  8. 1 kcal/mol 2 kcal/mol Ubiquitin-ubiquitin binding domains(UBDs) Validation: binding affinities Ub/CUE Ub/UIM1 Ub/UBA Ub/GAT Ub/DUIM Ub/UIM2 UbL/UBA UbL/UIM UbL/UIM2 Experiments Simulations (YCK, Hummer; J. Mol. Biol. 375, 1416, 2008)

  9. Vps27/Hse1(yeast) Hrs/STAM(human) Application I:multi-vesicular body (MVB) protein sorting machinery • The ESCRT machinery targets ubiquitinated transmembrane proteins for degradation in the lysosome or yeast vacuole • ESCRTs are required for HIV budding at the plasma membrane (Prag, Watson, YCK, Beach, Ghirlando, Hummer, Bonifacino, Hurley, Dev. Cell 12, 973, 2007)

  10. Structure of the assembled Vps27/Hse1 complex Vps27/Hse1 complex membrane (Prag, Watson, YCK, Beach, Ghirlando, Hummer, Bonifacino, Hurley, Dev. Cell 12, 973, 2007)

  11. Ubiquitin UIM Distance between Ub and UIM 80 60 Distance (Å) 40 20 Bound Unbound 0 1000 2000 3000 4000 5000 MC steps Radius of gyration 60 Rg UIM1 50 Globular protein: Rg = 30Å Ubiquitin 40 120 130 140 150 160 170 System size (Å) Vps27/Hse1 complex is dynamic and open

  12. Hrs/STAM(human) complex also shows open structures Experiments w/o random coil Restraint simulations w/ random coil Hrs/STAM Hydrodynamic radius Unrestraint simulations

  13. UIM1 binding Positive cooperativity enhances Vps27 binding to ubiquitin VHS UIM2 Ub FYVE Fraction of Vps27 bound to ubiquitin Vps27 binding membrane UIM1 (b) (d) FYVEdomain is tethered to the membrane Proteins can move freely parallel to the membrane Ubiquitin concentration [10-3/nm2] (YCK, Hummer; J. Mol. Biol. 375, 1416, 2008)

  14. Summary I: Simulations of Vps27/Hse1 • Dynamic and open structure • Important for targeting a variety of ubiquitinated cargos • Cooperative binding of ubiquitin via nonspecific interactions • Essential for function at low biological concentrations • Are nonspecific interactions detectable?

  15. Application II: Transient encounter complexes probed by simulation and NMR • Paramagnetic relaxation enhancement (PRE) probes the presence of low-population (<10%) transient encounter complexes Iwahara, Clore, Nature440, 1227, 2006 paramagnetic label(unpaired electron) proton spin probed PRE ~ 1/r 6

  16. NMR Paramagnetic Relaxation Enhancement (encounter complexes of HPr-IIAMannose?) • PRE of backbone amide protons on IIAMannose Mn2+ HPr IIAMannose Can we simulate encounter complexes? (Tang, Iwahara, Clore, Nature444, 383, 2006)

  17. Binding affinity Structure encounter complexes distance from specific structure >80% Kd(exp)~30 mM  error ~ kT ln 60 ~ 2.5kcal/mol ~ stereospecific complex Replica-exchange simulations of HPr-IIAMan complex • Coarse-grained simulation model (YCK, Hummer; J. Mol. Biol. 375, 1416, 2008) (YCK, Tang, Clore, Hummer, PNAS USA 105, 12855, 2008)

  18. PRE profiles of HPr-IIAMan complex • PRE of backbone amide protons on IIAMannose magnitude? (YCK, Tang, Clore, Hummer, Proc. Natl. Acad. Sci. USA 105, 12855, 2008)

  19. Reweighting of simulation structures • Simulation model should not be expected to produce accurate populations • 2 kT binding free energy difference → 10-fold difference in population • Cluster the structures of the specific and non-specific complexes • Re-weight the populations of the clusters to match PRE profiles distance from specific structure (YCK, Tang, Clore, Hummer, Proc. Natl. Acad. Sci. USA 105, 12855, 2008)

  20. PRE profiles of HPr-IIAMan complex • PRE of backbone amide protons on IIAMannose encounter complex Specific complex • Non-specific population <10% • Single dominant non-specific cluster • 110o rotation of stereospecific complex (YCK, Tang, Clore, Hummer, Proc. Natl. Acad. Sci. USA 105, 12855, 2008)

  21. Energy landscape of protein complex formation • Funnel-like energy landscape { Unstructurednon-specific { Structurednon-specific { Stereospecific (YCK, Tang, Clore, Hummer, Proc. Natl. Acad. Sci. USA 105, 12855, 2008)

  22. Summary II: Biology of transient encounter complexes • Accelerated on-rate (barnase: Schreiber, Fersht, Nat Struct Biol 3, 427, 1996) • Strengthening of weak specific interactions in multi-protein assemblies (Vps27) • Alternative binding modes (mannose transport: Hu et al. J Biol Chem 283, 11024, 2008) • Evolutionary remnants of earlier specific interactions?

  23. Conclusion • Coarse-grained model and transferable energy function provide valuable and complementary information regarding structures and dynamics of multi-protein assemblies and transient encounter complexes

  24. Acknowledgments • Gerhard Hummer (NIDDK, NIH) ESCRT complex • James Hurley (NIDDK, NIH) PRE of encounter complexes • G. Marius Clore (NIDDK, NIH) • Chun Tang (U. Missouri) Computational resources • NIH Biowulf • Helix Systems Staff

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