Effect of Ser392 phosphorylation on the structure and dynamics of the polybasic domain of ADP ribosylation factor nucleotide site opener protein: a molecular simulation study.

ADP ribosylation factor nucleotide site opener (ARNO) as a guanine nucleotide exchange factor (GEF) activates small GTPases called ADP ribosylation factors (Arfs), which function as molecular switches and regulate a variety of cell biological events. ARNO directly interacts with the transmembrane a2-subunit isoform of the proton-pumping vacuolar ATPase in an acidification-dependent manner, and this interaction plays a crucial role in the regulation of the protein degradation pathway. A recent study reported specific interactions of a2N with the ARNO375-400 peptide corresponding to the polybasic (PB) domain of ARNO, which is a crucial regulatory element in the autoregulation and modulation of Arf-GEF activity. Interestingly, phosphorylation of Ser392 completely abolishes this interaction, and the experimental structure shows significant structural rearrangements. To investigate the effect of Ser392 phosphorylation on the structure and dynamics of the ARNO375-400 peptide, we employed all atom molecular dynamics (MD) simulations of the phosphorylated and unphosphorylated PB domain of the ARNO protein. A Hamiltonian-based replica exchange method called biasing potential replica exchange MD was used to enhance conformational sampling. Simulations predicted that the isolated PB domain is highly flexible, with the C-terminal region of the unphosphorylated state being unstable. In contrast, Ser392 phosphorylation increases the overall stability of the peptide. In agreement with experimental results, our simulations further support the hypothesis that phosphorylation induces disorder to order transitions and provide new insights into the structural dynamics of the PB domain. Phosphorylation of Ser392 appears to stabilize the C-terminal α-helix via formation of salt bridges between phospho-Ser392 and Arg390, Lys395, and Lys396.

[1]  M. Zacharias,et al.  Folding of Trp-cage Mini Protein Using Temperature and Biasing Potential Replica—Exchange Molecular Dynamics Simulations , 2009, International journal of molecular sciences.

[2]  J. Missimer,et al.  Influence of 63Ser phosphorylation and dephosphorylation on the structure of the stathmin helical nucleation sequence: a molecular dynamics study. , 2012, Biochemistry.

[3]  Simon W. Ginzinger,et al.  SHIFTX2: significantly improved protein chemical shift prediction , 2011, Journal of biomolecular NMR.

[4]  D. Ausiello,et al.  V-ATPase interacts with ARNO and Arf6 in early endosomes and regulates the protein degradative pathway , 2006, Nature Cell Biology.

[5]  David D. Thomas,et al.  Molecular dynamics simulations reveal a disorder-to-order transition on phosphorylation of smooth muscle myosin. , 2007, Biophysical journal.

[6]  J. Casanova.  Regulation of Arf Activation: the Sec7 Family of Guanine Nucleotide Exchange Factors , 2007, Traffic.

[7]  P. Kollman,et al.  Settle: An analytical version of the SHAKE and RATTLE algorithm for rigid water models , 1992 .

[8]  Martin Zacharias,et al.  Folding simulations of Trp‐cage mini protein in explicit solvent using biasing potential replica‐exchange molecular dynamics simulations , 2009, Proteins.

[9]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

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

[11]  Michael Feig,et al.  MMTSB Tool Set: enhanced sampling and multiscale modeling methods for applications in structural biology. , 2004, Journal of molecular graphics & modelling.

[12]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[13]  Yuko Okamoto,et al.  Generalized-ensemble algorithms: enhanced sampling techniques for Monte Carlo and molecular dynamics simulations. , 2003, Journal of molecular graphics & modelling.

[14]  Frank McCormick,et al.  The GTPase superfamily: a conserved switch for diverse cell functions , 1990, Nature.

[15]  Smart Jl,et al.  PHOSPHORYLATION STABILIZES THE N-TERMINI OF ALPHA -HELICES , 1999 .

[16]  F. Richards,et al.  The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. , 1992, Biochemistry.

[17]  Martin Zacharias,et al.  Enhanced sampling of peptide and protein conformations using replica exchange simulations with a peptide backbone biasing‐potential , 2006, Proteins.

[18]  J. Casanova,et al.  Regulation of ARNO nucleotide exchange by a PH domain electrostatic switch , 1999, Current Biology.

[19]  W. Jahnke,et al.  Phosphorylation disrupts the central helix in Op18/stathmin and suppresses binding to tubulin , 2001, EMBO reports.

[20]  R. Klausner,et al.  ARF: a key regulatory switch in membrane traffic and organelle structure. , 1994, Current opinion in cell biology.

[21]  Anselm H. C. Horn,et al.  AMBER force-field parameters for phosphorylated amino acids in different protonation states: phosphoserine, phosphothreonine, phosphotyrosine, and phosphohistidine , 2006, Journal of molecular modeling.

[22]  Wei Zhang,et al.  A point‐charge force field for molecular mechanics simulations of proteins based on condensed‐phase quantum mechanical calculations , 2003, J. Comput. Chem..

[23]  Gianluigi Veglia,et al.  Serine 16 phosphorylation induces an order-to-disorder transition in monomeric phospholamban. , 2005, Biochemistry.

[24]  Crislyn D'Souza-Schorey,et al.  ARF proteins: roles in membrane traffic and beyond , 2006, Nature Reviews Molecular Cell Biology.

[25]  G. Grüber,et al.  Specific motifs of the V-ATPase a2-subunit isoform interact with catalytic and regulatory domains of ARNO. , 2010, Biochimica et biophysica acta.

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

[27]  V. Marshansky.  The V-ATPase a2-subunit as a putative endosomal pH-sensor. , 2007, Biochemical Society transactions.

[28]  Martin Zacharias,et al.  Efficient evaluation of sampling quality of molecular dynamics simulations by clustering of dihedral torsion angles and Sammon mapping , 2009, J. Comput. Chem..

[29]  David D. Thomas,et al.  The alpha-helical propensity of the cytoplasmic domain of phospholamban: a molecular dynamics simulation of the effect of phosphorylation and mutation. , 2005, Biophysical journal.

[30]  J. Rubinstein,et al.  The N Termini of a-Subunit Isoforms Are Involved in Signaling between Vacuolar H+-ATPase (V-ATPase) and Cytohesin-2* , 2013, The Journal of Biological Chemistry.

[31]  Srinivasaraghavan Kannan,et al.  Application of biasing‐potential replica‐exchange simulations for loop modeling and refinement of proteins in explicit solvent , 2010, Proteins.

[32]  David D. Thomas,et al.  Phosphorylation-dependent conformational switch in spin-labeled phospholamban bound to SERCA. , 2006, Journal of molecular biology.

[33]  David D. Thomas,et al.  Phosphorylation-induced structural changes in smooth muscle myosin regulatory light chain , 2009, Proceedings of the National Academy of Sciences.

[34]  D. Lambright,et al.  Structural basis and mechanism of autoregulation in 3-phosphoinositide-dependent Grp1 family Arf GTPase exchange factors. , 2007, Molecular cell.