Protein–protein binding is often associated with changes in protonation state

pKa values of ionizable residues have been calculated using the PROPKA method and structures of 75 protein–protein complexes and their corresponding free forms. These pKa values were used to compute changes in protonation state of individual residues, net changes in protonation state of the complex relative to the uncomplexed proteins, and the correction to a binding energy calculated assuming standard protonation states at pH 7. For each complex, two different structures for the uncomplexed form of the proteins were used: the X‐ray structures determined for the proteins in the absence of the other protein and the individual protein structures taken from the structure of the complex (referred to as unbound and bound structures, respectively). In 28 and 77% of the cases considered here, protein–protein binding is accompanied by a complete (>95%) or significant (>50%) change in protonation state of at least one residue using unbound structures. Furthermore, in 36 and 61% of the cases, protein–protein binding is accompanied by a complete or significant net change in protonation state of the complex relative to the separated monomers. Using bound structures, the corresponding values are 12, 51, 20, and 48%. Comparison to experimental data suggest that using unbound and bound structures lead to over‐ and underestimation of binding‐induced protonation state changes, respectively. Thus, we conclude that protein–protein binding is often associated with changes in protonation state of amino acid residues and with changes in the net protonation state of the proteins. The pH‐dependent correction to the binding energy contributes at least one order of magnitude to the binding constant in 45 and 23%, using unbound and bound structures, respectively. Proteins 2008. © 2007 Wiley‐Liss, Inc.

[2]  Gary D Bader,et al.  Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry , 2002, Nature.

[3]  S. Anderson,et al.  Ionizable P1 Residues in Serine Proteinase Inhibitors Undergo Large pK Shifts on Complex Formation (*) , 1995, The Journal of Biological Chemistry.

[4]  Sarah A. Teichmann,et al.  Principles of protein-protein interactions , 2002, ECCB.

[5]  J. Mccammon,et al.  Determination of the pKa values of titratable groups of an antigen-antibody complex, HyHEL-5-hen egg lysozyme. , 1995, Protein engineering.

[6]  B Tidor,et al.  Charge optimization leads to favorable electrostatic binding free energy. , 1999, Physical review. E, Statistical physics, plasmas, fluids, and related interdisciplinary topics.

[7]  Z. Weng,et al.  Protein–protein docking benchmark 2.0: An update , 2005, Proteins.

[8]  T. Clackson,et al.  A hot spot of binding energy in a hormone-receptor interface , 1995, Science.

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

[10]  Ruben Abagyan,et al.  Statistical analysis and prediction of protein–protein interfaces , 2005, Proteins.

[11]  K. Sharp,et al.  Electrostatic interactions in hirudin-thrombin binding. , 1996, Biophysical chemistry.

[12]  James R Horn,et al.  Structure and energetics of protein-protein interactions: the role of conformational heterogeneity in OMTKY3 binding to serine proteases. , 2003, Journal of molecular biology.

[13]  A. Elcock,et al.  Computer Simulation of Protein−Protein Interactions , 2001 .

[14]  Zhiping Weng,et al.  A protein–protein docking benchmark , 2003, Proteins.

[15]  Joanna Trylska,et al.  Thermodynamic linkage between the binding of protons and inhibitors to HIV‐1 protease , 2008, Protein science : a publication of the Protein Society.

[16]  M. Laskowski,et al.  NMR determination of pKa values for Asp, Glu, His, and Lys mutants at each variable contiguous enzyme-inhibitor contact position of the turkey ovomucoid third domain. , 2003, Biochemistry.

[17]  Mark Gerstein,et al.  Bridging structural biology and genomics: assessing protein interaction data with known complexes. , 2002, Drug discovery today.

[18]  R. Nussinov,et al.  Conservation of polar residues as hot spots at protein interfaces , 2000, Proteins.

[19]  B. Rost,et al.  Predicted protein–protein interaction sites from local sequence information , 2003, FEBS letters.

[20]  M. Karplus,et al.  Multiple-site titration curves of proteins: an analysis of exact and approximate methods for their calculation , 1991 .

[21]  J. Aqvist,et al.  A new method for predicting binding affinity in computer-aided drug design. , 1994, Protein engineering.

[22]  Arieh Warshel,et al.  The Reorganization Energy of Cytochrome c Revisited , 1997 .

[23]  J. Thornton,et al.  Structural characterisation and functional significance of transient protein-protein interactions. , 2003, Journal of molecular biology.

[24]  T. Harris,et al.  Structural Basis of Perturbed pKa Values of Catalytic Groups in Enzyme Active Sites , 2002, IUBMB life.

[25]  J A McCammon,et al.  Electrostatic contributions to the stability of halophilic proteins. , 1998, Journal of molecular biology.

[26]  K. P. Murphy,et al.  Dissecting the energetics of a protein-protein interaction: the binding of ovomucoid third domain to elastase. , 1997, Journal of molecular biology.

[27]  S. Wodak,et al.  Assessment of CAPRI predictions in rounds 3–5 shows progress in docking procedures , 2005, Proteins.

[28]  Emil Alexov,et al.  Electrostatic properties of protein-protein complexes. , 2006, Biophysical journal.

[29]  G. Ullmann,et al.  Relations between Protonation Constants and Titration Curves in Polyprotic Acids: A Critical View , 2003 .

[30]  Jan H. Jensen,et al.  The Prediction of Protein pKa's Using QM/MM: The pKa of Lysine 55 in Turkey Ovomucoid Third Domain , 2002 .

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

[32]  A. Warshel,et al.  Electrostatic contributions to protein–protein binding affinities: Application to Rap/Raf interaction , 1998, Proteins.

[33]  J. Mccammon,et al.  pH dependence of antibody/lysozyme complexation. , 1997, Biochemistry.

[34]  Barry Honig,et al.  On the role of electrostatic interactions in the design of protein-protein interfaces. , 2002, Journal of molecular biology.

[35]  D. Moss,et al.  Benchmarking pKa prediction , 2006, BMC Biochemistry.

[36]  J. Åqvist,et al.  Free energy calculations show that acidic P1 variants undergo large pKa shifts upon binding to trypsin , 2006, Proteins.

[37]  B Honig,et al.  On the pH dependence of protein stability. , 1993, Journal of molecular biology.

[38]  A. Warshel,et al.  Calculations of antibody-antigen interactions: microscopic and semi-microscopic evaluation of the free energies of binding of phosphorylcholine analogs to McPC603. , 1992, Protein engineering.

[39]  A. Velázquez‐Campoy,et al.  Thermodynamic dissection of the binding energetics of KNI‐272, a potent HIV‐1 protease inhibitor , 2000, Protein science : a publication of the Protein Society.

[40]  Huan-Xiang Zhou,et al.  Comparison of calculation and experiment implicates significant electrostatic contributions to the binding stability of barnase and barstar. , 2003, Biophysical journal.

[41]  W. Stites,et al.  Protein−Protein Interactions: Interface Structure, Binding Thermodynamics, and Mutational Analysis , 1997 .

[42]  G. Matthias Ullmann,et al.  Decomposing complex cooperative ligand binding into simple components: Connections between microscopic and macroscopic models , 2004 .

[43]  K. P. Murphy,et al.  Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry. , 1996, Biophysical journal.

[44]  S. Subramaniam,et al.  Continuum electrostatic methods applied to pH-dependent properties of antibody-antigen association. , 2000, Methods.