A fluid salt-bridging cluster and the stabilization of p53.

p53 is a homotetrameric tumor suppressor protein that is found to be mutated in most human cancers. Some of these mutations, particularly mutations to R337, fall in the tetramerization domain and cause defects in tetramer formation leading to loss of function. Mutation to His at this site has been found to destabilize the tetramer in a pH-dependent fashion. In structures of the tetramerization domain determined by crystallography, R337 from one monomer makes a salt bridge with D352 from another monomer, apparently helping to stabilize the tetramer. Here we present molecular dynamics simulations of wild-type p53 and the R337His mutant at several different pH and salt conditions. We find that the 337-352 salt bridge is joined by two other charged side chains, R333 and E349. These four residues do not settle into a fixed pattern of salt bridging, but continue to exchange salt-bridging partners on the nanosecond time scale throughout the simulation. This unusual system of fluid salt bridging may explain the previous finding from alanine scanning experiments that R333 contributes significantly to protein stability, even though in the crystal structure it is extended outward into solvent. This extended conformation of R333 appears to be the result of a specific crystal contact and, this contact being absent in the simulation, R333 turns inward to join its interaction partners. When R337 is mutated to His but remains positively charged, it maintains the original interaction with D352, but the newly observed interaction with E349 is weakened, accounting for the reduced stability of R337H even under mildly acidic conditions. When this His is deprotonated, the interaction with D352 is also lost, accounting for the further destabilization observed under mildly alkaline conditions. Simulations were carried out using both explicit and implicit solvent models, and both displayed similar behavior of the fluid salt-bridging cluster, suggesting that implicit solvent models can capture at least the qualitative features of this phenomenon as well as explicit solvent. Simulations under strongly acidic conditions in implicit solvent displayed the beginnings of the unfolding process, a destabilization of the hydrophobic dimer-dimer interface. Computational alanine scanning using the molecular mechanics Poisson-Boltzmann surface area method showed significant correlation to experimental unfolding data for charged and polar residues, but much weaker correlation for hydrophobic residues.

[1]  A. Gronenborn,et al.  Four p53 DNA-binding domain peptides bind natural p53-response elements and bend the DNA. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[2]  R. Kriwacki,et al.  Disruption of an intermonomer salt bridge in the p53 tetramerization domain results in an increased propensity to form amyloid fibrils , 2005, Protein science : a publication of the Protein Society.

[3]  G. Marius Clore,et al.  Refined solution structure of the oligomerization domain of the tumour suppressor p53 , 1995, Nature Structural Biology.

[4]  J. Garin,et al.  The in vitro phosphorylation of p53 by calcium-dependent protein kinase C--characterization of a protein-kinase-C-binding site on p53. , 1997, European journal of biochemistry.

[5]  Lennart Nilsson,et al.  Thermal unfolding simulations of a multimeric protein—Transition state and unfolding pathways , 2005, Proteins.

[6]  D. Case,et al.  Exploring protein native states and large‐scale conformational changes with a modified generalized born model , 2004, Proteins.

[7]  C. Arrowsmith,et al.  Thermodynamic analysis of the structural stability of the tetrameric oligomerization domain of p53 tumor suppressor. , 1995, Biochemistry.

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

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

[10]  M Karplus,et al.  Binding free energies and free energy components from molecular dynamics and Poisson-Boltzmann calculations. Application to amino acid recognition by aspartyl-tRNA synthetase. , 2001, Journal of molecular biology.

[11]  P. Chène,et al.  The role of tetramerization in p53 function , 2001, Oncogene.

[12]  Vijay S Pande,et al.  Kinetic computational alanine scanning: application to p53 oligomerization. , 2006, Journal of molecular biology.

[13]  P. Kollman,et al.  Continuum Solvent Studies of the Stability of DNA, RNA, and Phosphoramidate−DNA Helices , 1998 .

[14]  R. Ribeiro,et al.  Reversible amyloid formation by the p53 tetramerization domain and a cancer-associated mutant. , 2003, Journal of molecular biology.

[15]  A. Fersht,et al.  Nine hydrophobic side chains are key determinants of the thermodynamic stability and oligomerization status of tumour suppressor p53 tetramerization domain , 1998, The EMBO journal.

[16]  G. Wahl,et al.  A leucine‐rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking , 1999, The EMBO journal.

[17]  D. Case,et al.  Thermodynamics of a reverse turn motif. Solvent effects and side-chain packing. , 1997, Journal of molecular biology.

[18]  P. Kollman,et al.  How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? , 2000 .

[19]  C. Stratakis,et al.  An inherited p53 mutation that contributes in a tissue-specific manner to pediatric adrenal cortical carcinoma , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[20]  M. Grütter,et al.  Crystallization and structure solution of p53 (residues 326-356) by molecular replacement using an NMR model as template. , 1998, Acta crystallographica. Section D, Biological crystallography.

[21]  P. Beroza,et al.  Application of a pairwise generalized Born model to proteins and nucleic acids: inclusion of salt effects , 1999 .

[22]  Barry Honig,et al.  Extending the Applicability of the Nonlinear Poisson−Boltzmann Equation: Multiple Dielectric Constants and Multivalent Ions† , 2001 .

[23]  Vijay S Pande,et al.  Dimerization of the p53 oligomerization domain: identification of a folding nucleus by molecular dynamics simulations. , 2005, Journal of molecular biology.

[24]  M. G. Mateu,et al.  Hydrogen exchange of the tetramerization domain of the human tumour suppressor p53 probed by denaturants and temperature. , 2001, European journal of biochemistry.

[25]  Piotr Cieplak,et al.  Molecular dynamics and free energy analyses of cathepsin D-inhibitor interactions: insight into structure-based ligand design. , 2002, Journal of medicinal chemistry.

[26]  Adrian A Canutescu,et al.  Access the most recent version at doi: 10.1110/ps.03154503 References , 2003 .

[27]  Patrick W. Lee,et al.  Biogenesis of p53 Involves Cotranslational Dimerization of Monomers and Posttranslational Dimerization of Dimers , 2002, The Journal of Biological Chemistry.

[28]  R. Ribeiro,et al.  A novel mechanism of tumorigenesis involving pH-dependent destabilization of a mutant p53 tetramer , 2002, Nature Structural Biology.

[29]  M. Kubbutat,et al.  Regulation of Mdm2-Directed Degradation by the C Terminus of p53 , 1998, Molecular and Cellular Biology.

[30]  H. Dyson,et al.  Coupling of folding and binding for unstructured proteins. , 2002, Current opinion in structural biology.

[31]  B. Brooks,et al.  Langevin dynamics of peptides: The frictional dependence of isomerization rates of N‐acetylalanyl‐N′‐methylamide , 1992, Biopolymers.

[32]  P. Kollman,et al.  Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models. , 2000, Accounts of chemical research.

[33]  Alan R. Fersht,et al.  Mechanism of folding and assembly of a small tetrameric protein domain from tumor suppressor p53 , 1999, Nature Structural Biology.

[34]  A. Gronenborn,et al.  High-resolution structure of the oligomerization domain of p53 by multidimensional NMR. , 1994, Science.

[35]  C. Maki.  Oligomerization Is Required for p53 to be Efficiently Ubiquitinated by MDM2* , 1999, The Journal of Biological Chemistry.

[36]  C. Arrowsmith,et al.  Solution structure of the tetrameric minimum transforming domain of p53 , 1995, Nature Structural Biology.

[37]  Kohei Uosaki,et al.  Unfolding, aggregation, and amyloid formation by the tetramerization domain from mutant p53 associated with lung cancer. , 2006, Biochemistry.

[38]  Y Taya,et al.  The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites. , 2000, Genes & development.

[39]  P. Tompa.  Intrinsically unstructured proteins. , 2002, Trends in biochemical sciences.

[40]  N. Horikoshi,et al.  Blockage by Adenovirus E4orf6 of Transcriptional Activation by the p53 Tumor Suppressor , 1996, Science.

[41]  T. Yoshimoto,et al.  The relationship among p53 oligomer formation, structure and transcriptional activity using a comprehensive missense mutation library , 2005, Oncogene.

[42]  Markus Meuwly,et al.  Importance of individual side chains for the stability of a protein fold: Computational alanine scanning of the insulin monomer , 2006, J. Comput. Chem..

[43]  P. Kollman,et al.  Computational Alanine Scanning To Probe Protein−Protein Interactions: A Novel Approach To Evaluate Binding Free Energies , 1999 .

[44]  M. Hollstein,et al.  p53 and human cancer: the first ten thousand mutations. , 2000, Advances in cancer research.

[45]  Donald Bashford,et al.  Structural details, pathways, and energetics of unfolding apomyoglobin. , 2003, Journal of molecular biology.

[46]  D. Case,et al.  Insights into protein-protein binding by binding free energy calculation and free energy decomposition for the Ras-Raf and Ras-RalGDS complexes. , 2003, Journal of molecular biology.

[47]  B. Vogelstein,et al.  Participation of p53 protein in the cellular response to DNA damage. , 1991, Cancer research.

[48]  David A. Case,et al.  A computational study of the role of solvation effects in reverse turn formation in the tetrapeptides APGD and APGN , 1997 .

[49]  N. Pavletich,et al.  Crystal structure of the tetramerization domain of the p53 tumor suppressor at 1.7 angstroms , 1995, Science.

[50]  A. Fersht,et al.  Rescuing the function of mutant p53 , 2001, Nature Reviews Cancer.

[51]  P. Kollman,et al.  Use of MM-PBSA in reproducing the binding free energies to HIV-1 RT of TIBO derivatives and predicting the binding mode to HIV-1 RT of efavirenz by docking and MM-PBSA. , 2001, Journal of the American Chemical Society.

[52]  D. Lane,et al.  p53, guardian of the genome , 1992, Nature.

[53]  E. Flemington,et al.  The Epstein‐Barr virus bZIP transcription factor Zta causes G0/G1 cell cycle arrest through induction of cyclin‐dependent kinase inhibitors. , 1996, The EMBO journal.

[54]  Pedro Alexandrino Fernandes,et al.  Computational alanine scanning mutagenesis—An improved methodological approach , 2007, J. Comput. Chem..

[55]  A. Gronenborn,et al.  The oligomerization domain of p53: Crystal structure of the trigonal form , 1996, FEBS letters.

[56]  A. Roitberg,et al.  All-atom structure prediction and folding simulations of a stable protein. , 2002, Journal of the American Chemical Society.

[57]  C. Harris,et al.  The IARC TP53 database: New online mutation analysis and recommendations to users , 2002, Human mutation.

[58]  David E. Housman,et al.  Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours , 1996, Nature.

[59]  V. Zhurkin,et al.  p53-induced DNA bending and twisting: p53 tetramer binds on the outer side of a DNA loop and increases DNA twisting. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[60]  W. C. Still,et al.  Approximate atomic surfaces from linear combinations of pairwise overlaps (LCPO) , 1999 .

[61]  Emil Alexov,et al.  Rapid grid‐based construction of the molecular surface and the use of induced surface charge to calculate reaction field energies: Applications to the molecular systems and geometric objects , 2002, J. Comput. Chem..