Are hot-spots occluded from water?

Protein–protein interactions are the basis of many biological processes and are governed by focused regions with high binding affinities, the warm- and hot-spots. It was proposed that these regions are surrounded by areas with higher packing density leading to solvent exclusion around them – “the O-ring theory.” This important inference still lacks sufficient demonstration. We have used Molecular Dynamics (MD) simulations to investigate the validity of the O-ring theory in the context of the conformational flexibility of the proteins, which is critical for function, in general, and for interaction with water, in particular. The MD results were analyzed for a variety of solvent-accessible surface area (SASA) features, radial distribution functions (RDFs), protein–water distances, and water residence times. The measurement of the average solvent-accessible surface area features for the warm- and hot-spots and the null-spots, as well as data for corresponding RDFs, identify distinct properties for these two sets of residues. Warm- and hot-spots are found to be occluded from the solvent. However, it has to be borne in mind that water-mediated interactions have significant power to construct an extensive and strongly bonded interface. We observed that warm- and hot-spots tend to form hydrogen bond (H-bond) networks with water molecules that have an occupancy around 90%. This study provides strong evidence in support of the O-ring theory and the results show that hot-spots are indeed protected from the bulk solvent. Nevertheless, the warm- and hot-spots still make water-mediated contacts, which are also important for protein–protein binding.

[1]  Peter G Wolynes,et al.  Role of water mediated interactions in protein-protein recognition landscapes. , 2003, Journal of the American Chemical Society.

[2]  Xing-Ming Zhao,et al.  APIS: accurate prediction of hot spots in protein interfaces by combining protrusion index with solvent accessibility , 2010, BMC Bioinformatics.

[3]  M. Sternberg,et al.  Rapid refinement of protein interfaces incorporating solvation: application to the docking problem. , 1998, Journal of molecular biology.

[4]  K. Henrick,et al.  Inference of macromolecular assemblies from crystalline state. , 2007, Journal of molecular biology.

[5]  Pedro A Fernandes,et al.  Hot spots—A review of the protein–protein interface determinant amino‐acid residues , 2007, Proteins.

[6]  B. Lee,et al.  The interpretation of protein structures: estimation of static accessibility. , 1971, Journal of molecular biology.

[7]  T. Hynes,et al.  Crystal structures of bovine chymotrypsin and trypsin complexed to the inhibitor domain of alzheimer's amyloid β‐protein precursor (APPI) and basic pancreatic trypsin inhibitor (BPTI): Engineering of inhibitors with altered specificities , 1997, Protein science : a publication of the Protein Society.

[8]  Jinyan Li,et al.  ‘Double water exclusion’: a hypothesis refining the O-ring theory for the hot spots at protein interfaces , 2009, Bioinform..

[9]  W. Delano.  Unraveling hot spots in binding interfaces: progress and challenges. , 2002, Current opinion in structural biology.

[10]  Joël Janin.  Basic Principles of Protein– Protein Interaction , 2009 .

[11]  G Schreiber,et al.  Energetics of protein-protein interactions: analysis of the barnase-barstar interface by single mutations and double mutant cycles. , 1995, Journal of molecular biology.

[12]  A. Bogan,et al.  Anatomy of hot spots in protein interfaces. , 1998, Journal of molecular biology.

[13]  Jinyan Li,et al.  Geometrically centered region: A “wet” model of protein binding hot spots not excluding water molecules , 2010, Proteins.

[14]  Pinak Chakrabarti,et al.  Hydration of protein–protein interfaces , 2005, Proteins.

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

[16]  R Shapiro,et al.  Analysis of the interactions of human ribonuclease inhibitor with angiogenin and ribonuclease A by mutagenesis: importance of inhibitor residues inside versus outside the C-terminal "hot spot". , 2000, Journal of molecular biology.

[17]  P. Kollman,et al.  A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules , 1995 .

[18]  R Shapiro,et al.  Site-specific mutagenesis reveals differences in the structural bases for tight binding of RNase inhibitor to angiogenin and RNase A. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[19]  T. Bhat,et al.  Bound water molecules and conformational stabilization help mediate an antigen-antibody association. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[20]  Charles Eigenbrot,et al.  Crystal Structure at 1.7 Å Resolution of VEGF in Complex with Domain 2 of the Flt-1 Receptor , 1997, Cell.

[21]  J. Janin,et al.  Dissecting protein–protein recognition sites , 2002, Proteins.

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

[23]  Jinyan Li,et al.  Protein binding hot spots and the residue-residue pairing preference: a water exclusion perspective , 2010, BMC Bioinformatics.

[24]  Zhenhua Li,et al.  DBAC: A simple prediction method for protein binding hot spots based on burial levels and deeply buried atomic contacts , 2011, BMC Systems Biology.

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

[26]  Julie C. Mitchell,et al.  KFC2: A knowledge‐based hot spot prediction method based on interface solvation, atomic density, and plasticity features , 2011, Proteins.

[27]  Nathan A. Baker,et al.  PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations , 2004, Nucleic Acids Res..

[28]  C. Chothia,et al.  The structure of protein-protein recognition sites. , 1990, The Journal of biological chemistry.

[29]  K. Ramnarayan,et al.  Glu-96 of Basic Fibroblast Growth Factor Is Essential for High Affinity Receptor Binding , 1995, The Journal of Biological Chemistry.

[30]  I. Moreira,et al.  Unraveling the importance of protein-protein interaction: application of a computational alanine-scanning mutagenesis to the study of the IgG1 streptococcal protein G (C2 fragment) complex. , 2006, The journal of physical chemistry. B.

[31]  S. Vajda,et al.  Anchor residues in protein-protein interactions. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[32]  M. Pantoliano,et al.  Identification and concerted function of two receptor binding surfaces on basic fibroblast growth factor required for mitogenesis. , 1994, The Journal of biological chemistry.

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

[34]  Peter A. Kollman,et al.  Computational alanine scanning of the 1:1 human growth hormone–receptor complex , 2002, J. Comput. Chem..

[35]  Jan H. Jensen,et al.  PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical pKa Predictions. , 2011, Journal of chemical theory and computation.

[36]  Jan H. Jensen,et al.  Very fast empirical prediction and rationalization of protein pKa values , 2005, Proteins.

[37]  Mickey Kosloff,et al.  Integrating energy calculations with functional assays to decipher the specificity of G protein – RGS protein interactions , 2011 .

[38]  Irina S. Moreira,et al.  Protein–protein recognition: a computational mutagenesis study of the MDM2–P53 complex , 2008 .

[39]  Brandon S. Zerbe,et al.  Domain motion and interdomain hot spots in a multidomain enzyme , 2010, Protein science : a publication of the Protein Society.

[40]  E. Goldman,et al.  Analysis of binding interactions in an idiotope-antiidiotope protein-protein complex by double mutant cycles. , 1997, Biochemistry.

[41]  Irina S. Moreira,et al.  Hot spot computational identification: Application to the complex formed between the hen egg white lysozyme (HEL) and the antibody HyHEL-10† , 2007 .

[42]  Irina S. Moreira,et al.  Structural Determinants of a Typical Leucine-Rich Repeat Protein , 2013 .

[43]  Pinak Chakrabarti,et al.  PRICE (PRotein Interface Conservation and Energetics): a server for the analysis of protein–protein interfaces , 2011, Journal of Structural and Functional Genomics.

[44]  R. Poljak,et al.  Crystal structure of an Fv-Fv idiotope-anti-idiotope complex at 1.9 A resolution. , 1996, Journal of molecular biology.

[45]  Qian Liu,et al.  Propensity vectors of low‐ASA residue pairs in the distinction of protein interactions , 2010, Proteins.

[46]  S. Anderson,et al.  Alanine Point-Mutations in the Reactive Region of Bovine Pancreatic Trypsin Inhibitor: Effects on the Kinetics and Thermodynamics of Binding to β-Trypsin and α-Chymotrypsin† , 1996 .

[47]  G. Winter,et al.  The contribution of contact and non-contact residues of antibody in the affinity of binding to antigen. The interaction of mutant D1.3 antibodies with lysozyme. , 1993, Journal of molecular biology.

[48]  B. Keyt,et al.  Identification of Vascular Endothelial Growth Factor Determinants for Binding KDR and FLT-1 Receptors , 1996, The Journal of Biological Chemistry.

[49]  P. Chakrabarti,et al.  Conservation and relative importance of residues across protein-protein interfaces , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[50]  Jan H. Jensen,et al.  Very fast prediction and rationalization of pKa values for protein–ligand complexes , 2008, Proteins.

[51]  Ozlem Keskin,et al.  Identification of computational hot spots in protein interfaces: combining solvent accessibility and inter-residue potentials improves the accuracy , 2009, Bioinform..

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

[53]  Carlos J. Camacho,et al.  ANCHOR: a web server and database for analysis of protein–protein interaction binding pockets for drug discovery , 2010, Nucleic Acids Res..

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

[55]  J. Onuchic,et al.  Water mediation in protein folding and molecular recognition. , 2006, Annual review of biophysics and biomolecular structure.

[56]  T. Clackson,et al.  Structural and functional analysis of the 1:1 growth hormone:receptor complex reveals the molecular basis for receptor affinity. , 1998, Journal of molecular biology.

[57]  H W Hellinga,et al.  Dissection of the protein G B1 domain binding site for human IgG Fc fragment , 1999, Protein science : a publication of the Protein Society.

[58]  W. Wooster,et al.  Crystal structure of , 2005 .

[59]  Irina S. Moreira,et al.  Unravelling Hot Spots: a comprehensive computational mutagenesis study , 2006 .

[60]  Ozlem Keskin,et al.  HotSprint: database of computational hot spots in protein interfaces , 2007, Nucleic Acids Res..

[61]  G J Kleywegt,et al.  Crystal structure of the C2 fragment of streptococcal protein G in complex with the Fc domain of human IgG. , 1995, Structure.

[62]  Pedro A Fernandes,et al.  Hot spot occlusion from bulk water: a comprehensive study of the complex between the lysozyme HEL and the antibody FVD1.3. , 2007, The journal of physical chemistry. B.

[63]  J. Deisenhofer,et al.  A structural basis of the interactions between leucine-rich repeats and protein ligands , 1995, Nature.

[64]  Pedro A Fernandes,et al.  Backbone Importance for Protein-Protein Binding. , 2007, Journal of chemical theory and computation.

[65]  G Schreiber,et al.  Interaction of barnase with its polypeptide inhibitor barstar studied by protein engineering. , 1993, Biochemistry.

[66]  P A Fernandes,et al.  Detailed microscopic study of the full zipA:FtsZ interface , 2006, Proteins.

[67]  A. Fersht,et al.  Protein-protein recognition: crystal structural analysis of a barnase-barstar complex at 2.0-A resolution. , 1994, Biochemistry.

[68]  A. Grollman.  Site specific mutagenesis. , 1990, Progress in clinical and biological research.

[69]  Jie Liang,et al.  Protein-protein interactions: hot spots and structurally conserved residues often locate in complemented pockets that pre-organized in the unbound states: implications for docking. , 2004, Journal of molecular biology.

[70]  E. Goldman,et al.  A mutational analysis of the binding of two different proteins to the same antibody. , 1996, Biochemistry.

[71]  Doheon Lee,et al.  A feature-based approach to modeling protein–protein interaction hot spots , 2009, Nucleic acids research.

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

[73]  Kurt S. Thorn,et al.  ASEdb: a database of alanine mutations and their effects on the free energy of binding in protein interactions , 2001, Bioinform..

[74]  W. Delano,et al.  Convergent solutions to binding at a protein-protein interface. , 2000, Science.

[75]  C. Chothia,et al.  Principles of protein–protein recognition , 1975, Nature.

[76]  Evgeny B. Krissinel,et al.  Crystal contacts as nature's docking solutions , 2010, J. Comput. Chem..

[77]  Mainak Guharoy,et al.  Conserved residue clusters at protein-protein interfaces and their use in binding site identification , 2010, BMC Bioinformatics.

[78]  David E. Kim,et al.  Computational Alanine Scanning of Protein-Protein Interfaces , 2004, Science's STKE.

[79]  J. Schlessinger,et al.  Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. , 2000, Molecular cell.

[80]  Philip Ball,et al.  Water as an Active Constituent in Cell Biology , 2008 .

[81]  Yan Zhang,et al.  The bacterial cell‐division protein ZipA and its interaction with an FtsZ fragment revealed by X‐ray crystallography , 2001, The EMBO journal.