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Cited by 211Protein Structure and DynamicsAdvanced Physical and Chemical Molecular Interactionsthermodynamics and calorimetric analyses

Efficient Determination of Protein–Protein Standard Binding Free Energies from First Principles

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Gumbart, James C., Roux, Benoît, Chipot, Christophe·2013·Journal of Chemical Theory and Computation
DOI 10.1021/ct400273tPMC PMC3809040
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Abstract

Characterizing protein-protein association quantitatively has been a longstanding challenge for computer simulations. Here, a theoretical framework is put forth that addresses this challenge on the basis of detailed all-atom molecular dynamics simulations with explicit solvent. The proposed methodology relies upon independent potential of mean force (PMF) free-energy calculations carried out sequentially, wherein the biological objects are restrained in the conformation, position and orientation of the bound state, using adequately chosen biasing potentials. These restraints systematically narrow down the configurational entropy available to the system and effectively guarantee that the relevant network of interactions is properly sampled as the two proteins reversibly associate. Decomposition of the binding process into consecutive, well-delineated stages, for both the protein complex and the individual, unbound partners, offers a rigorous definition of the standard state, from which the absolute binding free energy can be determined. The method is applied to the difficult case of the extracellular ribonuclease barnase binding to its intracellular inhibitor barstar. The calculated binding free energy is -21.0 ± 1.4 kcal/mol, which compares well with the experimental value of -19.0 ± 0.2 kcal/mol. The relatively small statistical error reflects the precision and convergence afforded by the PMF-based simulation methodology. In addition to providing an accurate reproduction of the standard binding free energy, the proposed strategy offers a detailed picture of the protein-protein interface, illuminating the thermodynamic forces that underlie reversible association. The application of the present formal framework to barnase:barstar binding provides a foundation for tackling nearly any protein-protein complex.

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  • The protocol has so far been successfully used in calculating the binding free energy of a variety of protein-protein complexes, in good agreement with experimental data, including a homodimeric α-helical complex.

    “The protocol has so far been successfully used in calculating the binding free energy of a variety of protein–protein complexes, in good agreement with experimental data,54,62,64,65,67,73−76 including a homodimeric α-helical complex.69”

    From Assessment of the Topology and Oligomerisation States of Coiled Coils Using Metadynamics with Conformational Restraints (2025) · cites this paper as [62]

    Citation 69 is attached specifically to the homodimeric α-helical example, not to the preceding claim about a variety of complexes. The Abstract (page unavailable) says this paper applies the method to barnase binding its inhibitor barstar—two different proteins—and reports good agreement with experiment for that complex, not a homodimeric α-helical complex. (LLM, conf=0.88)

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  • Simulations without such restraints have been reported to fail to converge and to yield binding free energies inconsistent with experimental data.

    “Simulations without such restraints have been reported to fail to converge and to yield binding free energies inconsistent with experimental data.62,65,69”

    From Assessment of the Topology and Oligomerisation States of Coiled Coils Using Metadynamics with Conformational Restraints (2025) · cites this paper as [62]

    The Introduction reports that comparable simulations without interfacial side-chain restraints failed to converge. It does not report a binding free energy from those unrestrained simulations or say that such a result was inconsistent with experimental data. The Discussion’s mention of slightly discrepant theoretical and experimental estimates concerns the restrained calculations. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • While the path-based PMF approach appears more tractable, it is still difficult to obtain converged free energy estimates for large biomolecules with complex geometries.

    “While the path-based PMF approach appears more tractable, it is still difficult to obtain converged free energy estimates for large biomolecules with complex geometries.53,62−68”

    From Assessment of the Topology and Oligomerisation States of Coiled Coils Using Metadynamics with Conformational Restraints (2025) · cites this paper as [62]

  • Alchemical decoupling of a complete protein molecule will usually result in poor convergence due to large free energy changes.

    “While in theory an alchemical pathway could be constructed via decoupling a binding partner in a protein–protein complex from its environment in the bound and unbound states, the alchemical decoupling of a complete protein molecule will usually result in poor convergence61 due to large free energy changes.62”

    From Assessment of the Topology and Oligomerisation States of Coiled Coils Using Metadynamics with Conformational Restraints (2025) · cites this paper as [62]

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Research works printed but not linked: 49

  1. [1]Chothia C Janin J Principles of protein-protein recognition Nature 1975 256 705 708 1153006
  2. [2]Jones S Thornton JM Principles of protein-protein interactions Proc. Natl. Acad. Sci. USA 1996 93 13 20 8552589 PMC40170
  3. [3]Stites WE Protein-protein interactions: Interface structure, binding thermodynamics, and mutational analysis Chem. Rev 1997 97 1233 1250 11851449
  4. [4]Angers S Salahpour A Bouvier M Dimerization: An emerging concept for G protein–coupled receptor ontogeny and function Annu. Rev. Pharmacol. Toxicol 2002 42 409 435 11807178
  5. [6]Vojtek AB Hollenberg SM Cooper JA Mammalian Ras interacts directly with the serine/threonine kinase Raf Cell 1993 74 205 214 8334704
  6. [7]Kortemme T Baker D A simple physical model for binding energy hot spots in protein-protein complexes Proc. Natl. Acad. Sci. USA 2002 99 14116 14121 12381794 PMC137846
  7. [8]Wang T Tomic S Gabdoulline RR Wade RC How optimal are the binding energetics of barnase and barstar? Biophys. J 2004 87 1618 1630 15345541 PMC1304567
  8. [9]Kim YC Tang C Clore GM Hummer G Replica exchange simulations of transient encounter complexes in protein-protein association Proc. Natl. Acad. Sci. USA 2008 105 12855 12860 18728193 PMC2529066
  9. [10]Periole X Knepp AM Sakmar TP Marrink SJ Huber T Structural determinants of the supramolecular organization of G protein-coupled receptors in bilayers J. Am. Chem. Soc 2012 134 10959 10965 22679925 PMC3406292
  10. [11]Gohlke H Kiel C Case DA Insights into protein-protein binding by binding free energy calculation and free energy decomposition for the Ras-Raf and Ras-RalGDS complexes J. Mol. Biol 2003 330 891 913 12850155
  11. [15]Chipot C Pohorille A Free energy calculations. Theory and applications in chemistry and biology 2007 Springer Verlag
  12. [16]Deng Y Roux B Computations of standard binding free energies with molecular dynamics simulations J. Phys. Chem. B 2009 113 2234 2246 19146384 PMC3837708
  13. [17]Gallicchio E Levy RM Advances in all atom sampling methods for modeling protein-ligand binding affinities Curr. Opin. Struct. Biol 2011 21 161 166 21339062 PMC3070828
  14. [18]Schreiber G Fersht AR The interaction of barnase with its polypeptide inhibitor barstar studied by protein engineering Biochemistry 1993 32 5145 5150 8494892
  15. [19]Shoup D Szabo A Role of diffusion in ligand binding to macromolecules and cell–bound receptors Biophys. J 1982 40 33 39 7139033 PMC1328970
  16. [21]Woo HJ Roux B Calculation of absolute protein–ligand binding free energy from computer simulations Proc. Natl. Acad. Sci. USA 2005 102 6825 6830 15867154 PMC1100764
  17. [22]Gan W Roux B Binding specificity of SH2 domains: insight from free energy simulations Proteins 2009 74 996 1007 18767163 PMC2635922
  18. [24]Dadarlat VM Skeel RD Dual role of protein phosphorylation in DNA activator/coactivator binding Biophys. J 2011 100 469 477 21244843 PMC3021674
  19. [25]Lau AY Roux B The hidden energetics of ligand binding and activation in a glutamate receptor Nat. Struct. Mol. Biol 2011 18 283 287 21317895 PMC3075596
  20. [26]Hartley RW Barnase and barstar: two small proteins to fold and fit together Trends Biochem. Sci 1989 14 450 454 2696173
  21. [27]Buckle AM Schreiber G Fersht AR Protein-protein recognition: Crystal structural analysis of a barnase-barstar complex at 2.0-Å resolution Biochemistry 1994 33 8878 8889 8043575
  22. [28]Schreiber G Fersht AR Rapid, electrostatically assisted association of proteins Nat. Struct. Biol 1996 3 427 431 8612072
  23. [29]Deng Y Roux B Calculation of standard binding free energies: Aromatic molecules in the T4 lysozyme L99A mutant J. Chem. Theory Comput 2006 2 1255 1273 26626834
  24. [30]Phillips JC Braun R Wang W Gumbart J Tajkhorshid E Villa E Chipot C Skeel RD Kale L Schulten K Scalable molecular dynamics with NAMD J. Comp. Chem 2005 26 1781 1802 16222654 PMC2486339
  25. [31]Best RB Zhu X Shim J Lopes PE Mittal J Feig M MacKerell AD Jr Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone ϕ, ψ and side-chain χ1 and χ2 dihedral angles J. Chem. Theory Comput 2012 8 3257 3273 23341755 PMC3549273
  26. [33]Hénin J Chipot C Overcoming free energy barriers using unconstrained molecular dynamics simulations J. Chem. Phys 2004 121 2904 2914 15291601
  27. [34]Hénin J Forin G Chipot C Klein ML Exploring multidimensional free energy landscapes using time-dependent biases on collective variables J. Chem. Theory Comput 2010 6 35 47 PMC12908704 26614317
  28. [37]Sotomayor M Schulten K Single-molecule experiments in vitro and in silico Science 2007 316 1144 1148 17525328
  29. [38]Dror RO Pan AC Arlow DH Borhani DW Maragakis P Shan Y Xu H Shaw DE Pathway and mechanism of drug binding to G-protein-coupled receptors Proc. Natl. Acad. Sci. USA 2011 108 13118 13123 21778406 PMC3156183
  30. [39]Spaar A Dammer C Gabdoulline RR Wade RC Helms V Diffusional encounter of barnase and barstar Biophys. J 2006 90 1913 1924 16361332 PMC1386772
  31. [40]Schreiber G Fersht AR Energetics of protein-protein interactions: Analysis of the barnase-barstar interface by single mutations and double mutant cycles J. Mol. Biol 1995 248 478 486 7739054
  32. [41]Rodriguez-Gomez D Darve E Pohorille A Assessing the efficiency of free energy calculation methods J. Chem. Phys 2004 120 3563 3578 15268518
  33. [42]Zhu F Hummer G Convergence and error estimation in free energy calculations using the weighted histogram analysis method J. Comp. Chem 2012 33 453 465 22109354 PMC3271861
  34. [43]Hartley RW Directed mutagenesis and barnase-barstar recognition Biochemistry 1993 32 5978 5984 8507637
  35. [44]Schreiber G Buckle AM Fersht AR Stability versus function: Two competing forces in the evolution of barstar Structure 1994 2 945 951 7866746
  36. [46]Cheng YK Rossky PJ Surface topography dependence of biomolecular hydrophobic hydration Nature 1998 392 696 699 9565030
  37. [48]ten Wolde PR Chandler D Drying-induced hydrophobic polymer collapse Proc. Natl. Acad. Sci. USA 2002 99 6539 6543 11983853 PMC124438
  38. [49]Liu P Huang X Zhou R Berne BJ Observation of a dewetting transition in the collapse of the melittin tetramer Nature 2005 437 159 162 16136146
  39. [50]Frisch C Schreiber G Johnson CM Fersht AR Thermodynamics of the interaction of barnase and barstar: changes in free energy versus changes in enthalpy on mutation J. Mol. Biol 1997 267 696 706 9126847
  40. [51]Lee LP Tidor B Barstar is electrostatically optimized for tight binding to barnase Nat. Struct. Biol 2001 8 73 76 11135675
  41. [52]Wang L Siu SWI Gu W Helms V Downhill binding energy surface of the barnase-barstar complex Biopolymers 2010 93 977 985 20540151
  42. [53]Tidor B Karplus M The contribution of vibrational entropy to molecular association The dimerization of insulin J. Mol. Biol 1994 238 405 414 8176732
  43. [54]Maragliano L Fischer A Vanden-Eijnden E Ciccotti G String method in collective variables: minimum free energy paths and isocommittor surfaces J. Chem. Phys 2006 125 24106 16848576
  44. [56]Hendsch ZS Tidor B Do salt bridges stabilize proteins? A continuum electrostatic analysis Protein Sci 1994 3 211 226 8003958 PMC2142793
  45. [57]Wimley WC White SH Experimentally determined hydrophobicity scale for proteins at membrane interfaces Nature Struct. Biol 1996 3 842 848 8836100
  46. [58]Lee LP Tidor B Optimization of binding electrostatics: Charge complementarity in the barnase-barstar protein complex Protein Sci 2001 10 362 377 11266622 PMC2373948
  47. [60]Mossakowska DE Nyberg K Fersht AR Kinetic characterization of the recombinant ribonuclease from Bacillus amyloliquefaciens (barnase) and investigation of key residues in catalysis by site-directed mutagenesis Biochemistry 1989 28 3843 3850 2665810
  48. [61]Kuttner YY Kozer N Segal E Schreiber G Haran G Separating the contribution of translational and rotational diffusion to protein association J. Am. Chem. Soc 2005 127 15138 15144 16248654
  49. [62]Shaw DE Deneroff MM Dror RO Kuskin JS Larson RH Salmon JK Young C Batson B Bowers KJ Chao JC Eastwood MP Gagliardo J Grossman JP Ho CR Ierardi DJ Kolossváry I Klepeis JL Layman T McLeavey C Moraes MA Mueller R Priest EC Shan Y Spengler J Theobald M Towles B Wang SC Anton, a special-purpose machine for molecular dynamics simulation Comm. ACM 2008 51 91 97

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Authors
Gumbart, J. C., Roux, B., & Chipot, C.
Affiliations
  • Georgia Institute of Technology (US)
  • Department of Biochemistry and Molecular Biology and Gordon Center for Integrative Science, The University of Chicago, Chicago, Illinois 60637, United States
  • Centre National de la Recherche Scientifique (FR)
Corresponding author
Chipot, Christophe
ORCID
Venue
Journal of Chemical Theory and Computation
Publisher
American Chemical Society (ACS)
Published
2013-08-01
Volume
9
Issue
8
Pages
3789-3798
ISSN
  • 1549-9618 (ISSN-L)
  • 1549-9626
DOI
10.1021/ct400273t
PMC
PMC3809040
OpenAlex
W2001687061
Cited by
211
References
3
Citations by year
2026: 8 · 2025: 12 · 2024: 15 · 2023: 16 · 2022: 28 · 2021: 12 · 2020: 17 · 2019: 19 · 2018: 21 · 2017: 14 · 2016: 21 · 2015: 7 · 2014: 17 · 2013: 4
FWCI
7.32
Type
journal article
Language
en
Open access
Locations
  • Journal of Chemical Theory and Computation (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
Funders
  • National Cancer Institute (R01CA093577)
  • National Institute of General Medical Sciences (RC2GM093307)
  • National Institute of Allergy and Infectious Diseases (K22AI100927)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Physical Sciences › Chemistry › Physical and Theoretical Chemistry › Advanced Physical and Chemical Molecular Interactions
  • Physical Sciences › Chemistry › Physical and Theoretical Chemistry › thermodynamics and calorimetric analyses
Keywords
protein-protein binding, binding free energy, potential of mean force, free energy decomposition, restrained molecular dynamics, biasing potential, standard states, barnase, barstar, ribonuclease inhibitor, protein-protein interfaces, conformational entropy

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