Molecular recognition of RAS/RAF complex at the membrane: Role of RAF cysteine-rich domain

Activation of RAF kinase involves the association of its RAS-binding domain (RBD) and cysteine-rich domain (CRD) with membrane-anchored RAS. However, the overall architecture of the RAS/RBD/CRD ternary complex and the orientations of its constituent domains at the membrane remain unclear. Here, we have combined all-atom and coarse-grained molecular dynamics (MD) simulations with experimental data to construct and validate a model of membrane-anchored CRD, and used this as a basis to explore models of membrane-anchored RAS/RBD/CRD complex. First, simulations of the CRD revealed that it anchors to the membrane via insertion of its two hydrophobic loops, which is consistent with our NMR measurements of CRD bound to nanodiscs. Simulations of the CRD in the context of membrane-anchored RAS/RBD then show how CRD association with either RAS or RBD could play an unexpected role in guiding the membrane orientations of RAS/RBD. This finding has implications for the formation of RAS-RAS dimers, as different membrane orientations of RAS expose distinct putative dimerization interfaces.

[1]  Berk Hess,et al.  GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers , 2015 .

[2]  R. Thapar,et al.  NMR characterization of full-length farnesylated and non-farnesylated H-Ras and its implications for Raf activation. , 2004, Journal of molecular biology.

[3]  Alfred Wittinghofer,et al.  Quantitative Analysis of the Complex between p21 and the Ras-binding Domain of the Human Raf-1 Protein Kinase (*) , 1995, The Journal of Biological Chemistry.

[4]  Wei Chen,et al.  Computational and biochemical characterization of two partially overlapping interfaces and multiple weak-affinity K-Ras dimers , 2017, Scientific Reports.

[5]  K. Berndt,et al.  Structure, dynamics and electrostatics of the active site of glutaredoxin 3 from Escherichia coli: comparison with functionally related proteins. , 2001, Journal of molecular biology.

[6]  John C Hunter,et al.  In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C , 2014, Proceedings of the National Academy of Sciences.

[7]  Sandrine Roy,et al.  Activity of Plasma Membrane-recruited Raf-1 Is Regulated by Ras via the Raf Zinc Finger* , 1997, The Journal of Biological Chemistry.

[8]  W. L. Jorgensen,et al.  Comparison of simple potential functions for simulating liquid water , 1983 .

[9]  H. Waldmann,et al.  The role of G-domain orientation and nucleotide state on the Ras isoform-specific membrane interaction , 2012, European Biophysics Journal.

[10]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[11]  D. Morrison,et al.  The complexity of Raf-1 regulation. , 1997, Current opinion in cell biology.

[12]  Erwin Laure,et al.  Solving Software Challenges for Exascale , 2014, Lecture Notes in Computer Science.

[13]  J. Åqvist,et al.  Molecular Dynamics Simulations of Water and Biomolecules with a Monte Carlo Constant Pressure Algorithm , 2004 .

[14]  Ross C. Walker,et al.  An overview of the Amber biomolecular simulation package , 2013 .

[15]  G. Voth,et al.  Interactions of Protein Kinase C-α C1A and C1B Domains with Membranes: A Combined Computational and Experimental Study , 2014, Journal of the American Chemical Society.

[16]  T. Darden,et al.  Particle mesh Ewald: An N⋅log(N) method for Ewald sums in large systems , 1993 .

[17]  S. Wiese,et al.  B- and C-RAF Display Essential Differences in Their Binding to Ras , 2007, Journal of Biological Chemistry.

[18]  Xavier Periole,et al.  Combining an Elastic Network With a Coarse-Grained Molecular Force Field: Structure, Dynamics, and Intermolecular Recognition. , 2009, Journal of chemical theory and computation.

[19]  Mu-Ping Nieh,et al.  Fluid phase lipid areas and bilayer thicknesses of commonly used phosphatidylcholines as a function of temperature. , 2011, Biochimica et biophysica acta.

[20]  J. Hurley,et al.  Crystal structure of the Cys2 activator-binding domain of protein kinase Cδ in complex with phorbol ester , 1995, Cell.

[21]  C. Der,et al.  RAS isoforms and mutations in cancer at a glance , 2016, Journal of Cell Science.

[22]  T. Kataoka,et al.  The Strength of Interaction at the Raf Cysteine-Rich Domain Is a Critical Determinant of Response of Raf to Ras Family Small GTPases , 1999, Molecular and Cellular Biology.

[23]  Helgi I. Ingólfsson,et al.  Martini straight: Boosting performance using a shorter cutoff and GPUs , 2016, Comput. Phys. Commun..

[24]  D. Barford,et al.  Wild-type and mutant B-RAF activate C-RAF through distinct mechanisms involving heterodimerization. , 2005, Molecular cell.

[25]  W. Kolch.  Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. , 2000, The Biochemical journal.

[26]  G. Wagner,et al.  Optimized phospholipid bilayer nanodiscs facilitate high-resolution structure determination of membrane proteins. , 2013, Journal of the American Chemical Society.

[27]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[28]  Taehoon Kim,et al.  CHARMM‐GUI: A web‐based graphical user interface for CHARMM , 2008, J. Comput. Chem..

[29]  Matthias Buck,et al.  Computational Modeling Reveals that Signaling Lipids Modulate the Orientation of K-Ras4A at the Membrane Reflecting Protein Topology. , 2017, Structure.

[30]  J. Avruch,et al.  An intact Raf zinc finger is required for optimal binding to processed Ras and for ras-dependent Raf activation in situ , 1997, Molecular and cellular biology.

[31]  C. Pritchard,et al.  Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells , 1995, Molecular and cellular biology.

[32]  M. Medkova,et al.  Interplay of C1 and C2 Domains of Protein Kinase C-α in Its Membrane Binding and Activation* , 1999, The Journal of Biological Chemistry.

[33]  M. Baccarini,et al.  Raf kinases in cancer–roles and therapeutic opportunities , 2011, Oncogene.

[34]  J. Colicelli,et al.  Human RAS Superfamily Proteins and Related GTPases , 2004, Science's STKE.

[35]  Ruth Nussinov,et al.  The higher level of complexity of K‐Ras4B activation at the membrane , 2016, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[36]  C. Der,et al.  Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer , 2007, Oncogene.

[37]  J. O’Leary,et al.  BRAFV600E: Implications for Carcinogenesis and Molecular Therapy , 2011, Molecular Cancer Therapeutics.

[38]  J. Reis-Filho,et al.  Kinase-Dead BRAF and Oncogenic RAS Cooperate to Drive Tumor Progression through CRAF , 2010, Cell.

[39]  C. Der,et al.  Elucidation of Binding Determinants and Functional Consequences of Ras/Raf-Cysteine-rich Domain Interactions* , 2000, The Journal of Biological Chemistry.

[40]  W. Xie,et al.  The cysteine-rich region of raf-1 kinase contains zinc, translocates to liposomes, and is adjacent to a segment that binds GTP-ras. , 1994, The Journal of biological chemistry.

[41]  D. Tieleman,et al.  Molecular dynamics simulation of a palmitoyl-oleoyl phosphatidylserine bilayer with Na+ counterions and NaCl. , 2004, Biophysical journal.

[42]  R. Nussinov,et al.  Allosteric effects of the oncogenic RasQ61L mutant on Raf-RBD. , 2015, Structure.

[43]  K. Dutta,et al.  A Small Molecule RAS-Mimetic Disrupts RAS Association with Effector Proteins to Block Signaling , 2016, Cell.

[44]  B. Brooks,et al.  An analysis of the accuracy of Langevin and molecular dynamics algorithms , 1988 .

[45]  J. Groves,et al.  K-Ras4B Remains Monomeric on Membranes over a Wide Range of Surface Densities and Lipid Compositions. , 2018, Biophysical journal.

[46]  Woonghee Lee,et al.  NMRFAM-SPARKY: enhanced software for biomolecular NMR spectroscopy , 2014, Bioinform..

[47]  D. Tieleman,et al.  The MARTINI force field: coarse grained model for biomolecular simulations. , 2007, The journal of physical chemistry. B.

[48]  C. Der,et al.  Two Distinct Raf Domains Mediate Interaction with Ras (*) , 1995, The Journal of Biological Chemistry.

[49]  Sandor Vajda,et al.  ClusPro: an automated docking and discrimination method for the prediction of protein complexes , 2004, Bioinform..

[50]  Marc Therrien,et al.  Crystal structure of a BRAF kinase domain monomer explains basis for allosteric regulation , 2014, Nature Structural &Molecular Biology.

[51]  S. Brown,et al.  NMR structure of a protein kinase C-gamma phorbol-binding domain and study of protein-lipid micelle interactions. , 1997, Biochemistry.

[52]  Gudrun Horn,et al.  Differential Interaction of the Ras Family GTP-binding Proteins H-Ras, Rap1A, and R-Ras with the Putative Effector Molecules Raf Kinase and Ral-Guanine Nucleotide Exchange Factor , 1996, The Journal of Biological Chemistry.

[53]  G. Ciccotti,et al.  Numerical Integration of the Cartesian Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes , 1977 .

[54]  R. Ghirlando,et al.  Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ , 2016, Proceedings of the National Academy of Sciences.

[55]  D. Tieleman,et al.  Perspective on the Martini model. , 2013, Chemical Society reviews.

[56]  S. Sligar,et al.  Chapter 11 - Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. , 2009, Methods in enzymology.

[57]  M. Parrinello,et al.  Canonical sampling through velocity rescaling. , 2007, The Journal of chemical physics.

[58]  Mitsuhiko Ikura,et al.  Oncogenic and RASopathy-associated K-RAS mutations relieve membrane-dependent occlusion of the effector-binding site , 2015, Proceedings of the National Academy of Sciences.

[59]  H. Berendsen,et al.  Molecular dynamics with coupling to an external bath , 1984 .

[60]  Priyanka Prakash,et al.  Oncogenic K-Ras Binds to an Anionic Membrane in Two Distinct Orientations: A Molecular Dynamics Analysis. , 2016, Biophysical journal.

[61]  P. Alexander,et al.  Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions , 2015, Scientific Reports.

[62]  Siewert J Marrink,et al.  Going Backward: A Flexible Geometric Approach to Reverse Transformation from Coarse Grained to Atomistic Models. , 2014, Journal of chemical theory and computation.

[63]  Alexander D. MacKerell,et al.  Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ(1) and χ(2) dihedral angles. , 2012, Journal of chemical theory and computation.

[64]  Richard Marais,et al.  The RAF proteins take centre stage , 2004, Nature Reviews Molecular Cell Biology.

[65]  D. Esposito,et al.  Dragging ras back in the ring. , 2014, Cancer cell.

[66]  S. Nosé,et al.  Constant pressure molecular dynamics for molecular systems , 1983 .

[67]  Sunhwan Jo,et al.  CHARMM‐GUI Membrane Builder toward realistic biological membrane simulations , 2014, J. Comput. Chem..

[68]  Wilfred F. van Gunsteren,et al.  A generalized reaction field method for molecular dynamics simulations , 1995 .

[69]  Alexander D. MacKerell,et al.  Update of the CHARMM all-atom additive force field for lipids: validation on six lipid types. , 2010, The journal of physical chemistry. B.

[70]  Sunhwan Jo,et al.  An ensemble dynamics approach to decipher solid-state NMR observables of membrane proteins. , 2012, Biochimica et biophysica acta.

[71]  Helgi I. Ingólfsson,et al.  Computational Lipidomics with insane: A Versatile Tool for Generating Custom Membranes for Molecular Simulations. , 2015, Journal of chemical theory and computation.

[72]  M. Therrien,et al.  Regulation of RAF protein kinases in ERK signalling , 2015, Nature Reviews Molecular Cell Biology.

[73]  F. Studier,et al.  Protein production by auto-induction in high density shaking cultures. , 2005, Protein expression and purification.

[74]  P. Hünenberger,et al.  Alternative schemes for the inclusion of a reaction-field correction into molecular dynamics simulations: Influence on the simulated energetic, structural, and dielectric properties of liquid water , 1998 .

[75]  Ozlem Keskin,et al.  GTP-Dependent K-Ras Dimerization. , 2015, Structure.

[76]  M. Medkova,et al.  Interplay of C1 and C2 domains of protein kinase C-alpha in its membrane binding and activation. , 1999, The Journal of biological chemistry.

[77]  Dima Kozakov,et al.  The ClusPro web server for protein–protein docking , 2017, Nature Protocols.

[78]  Krister Wennerberg,et al.  The Ras superfamily at a glance , 2005, Journal of Cell Science.

[79]  Susan S. Taylor,et al.  Allosteric Activation of Functionally Asymmetric RAF Kinase Dimers , 2013, Cell.

[80]  Berk Hess,et al.  Improving efficiency of large time‐scale molecular dynamics simulations of hydrogen‐rich systems , 1999, Journal of computational chemistry.

[81]  S. Yokoyama,et al.  Cysteine-rich Region of Raf-1 Interacts with Activator Domain of Post-translationally Modified Ha-Ras (*) , 1995, The Journal of Biological Chemistry.

[82]  H. Mott,et al.  The solution structure of the Raf-1 cysteine-rich domain: a novel ras and phospholipid binding site. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[83]  D. Barford,et al.  Mechanism of Activation of the RAF-ERK Signaling Pathway by Oncogenic Mutations of B-RAF , 2004, Cell.

[84]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..