On the role of electrostatic interactions in the design of protein-protein interfaces

Here, the methods of continuum electrostatics are used to investigate the contribution of electrostatic interactions to the binding of four protein-protein complexes; barnase-barstar, human growth hormone and its receptor, subtype N9 influenza virus neuraminidase and the NC41 antibody, the Ras binding domain (RBD) of kinase cRaf and a Ras homologue Rap1A. In two of the four complexes electrostatics are found to strongly oppose binding (hormone-receptor and neuraminidase-antibody complexes), in one case the net effect is close to zero (barnase-barstar) and in one case electrostatics provides a significant driving force favoring binding (RBD-Rap1A). In order to help understand the wide range of electrostatic contributions that were calculated, the electrostatic free energy was partitioned into contributions of individual charged and polar residues, salt bridges and networks involving salt bridges and hydrogen bonds. Although there is no one structural feature that accounts for the differences between the four interfaces, the extent to which the desolvation of buried charges is compensated by the formation of hydrogen bonds and ion pairs appears to be an important factor. Structural features that are correlated with contribution of an individual residue to stability are also discussed. These include partial burial of a charged group in the free monomer, the formation of networks involving charged and polar amino acids, and the formation of partially exposed ion-pairs. The total electrostatic contribution to binding is found to be inversely correlated with buried total and non-polar surface area. This suggests that different interfaces can be designed to exploit electrostatic and hydrophobic forces in very different ways.     

Cloud-point temperatures for lysozyme in electrolyte solutions: effect of salt type, salt concentration and pH

Liquid-liquid phase-separation data were obtained for aqueous saline solutions of hen egg-white lysozyme at a fixed protein concentration (87 g/l). The cloud-point temperature (CPT) was measured as a function of salt type and salt concentration to 3 M, at pH 4.0 and 7.0. Salts used included those from mono and divalent cations and anions. For the monovalent cations studied, as salt concentration increases, the CPT increases. For divalent cations, as salt concentration rises, a maximum in the CPT is observed and attributed to ion binding to the protein surface and subsequent water structuring. Trends for sulfate salts were dramatically different from those for other salts because sulfate ion is strongly hydrated and excluded from the lysozyme surface. For anions at fixed salt concentration, the CPT decreases with rising anion kosmotropic character. Comparison of CPTs for pH 4.0 and 7.0 revealed two trends. At low ionic strength for a given salt, differences in CPT can be explained in terms of repulsive electrostatic interactions between protein molecules, while at higher ionic strength, differences can be attributed to hydration forces. A model is proposed for the correlation and prediction of the CPT as a function of salt type and salt concentration. NaCl was chosen as a reference salt, and CPT deviations from that of NaCl were attributed to hydration forces. The Random Phase Approximation, in conjunction with a square-well potential, was used to calculate the strength of protein-protein interactions as a function of solution conditions for all salts studied.     

Electrostatic aspects of protein-protein interactions

Structural and mutational analyses reveal a central role for electrostatic interactions in protein-protein association. Experiment and theory both demonstrate that clusters of charged and polar residues that are located on protein-protein interfaces may enhance complex stability, although the total effect of electrostatics is generally net destabilizing. The past year also witnessed significant progress in our understanding of the effect of electrostatics on protein association kinetics, specifically in the characterization of a partially desolvated encounter complex.     

Electrostatic contributions to the binding free energy of the lambdacI repressor to DNA.

A model based on the nonlinear Poisson-Boltzmann (NLPB) equation is used to study the electrostatic contribution to the binding free energy of the lambdacI repressor to its operator DNA. In particular, we use the Poisson-Boltzmann model to calculate the pKa shift of individual ionizable amino acids upon binding. We find that three residues on each monomer, Glu34, Glu83, and the amino terminus, have significant changes in their pKa and titrate between pH 4 and 9. This information is then used to calculate the pH dependence of the binding free energy. We find that the calculated pH dependence of binding accurately reproduces the available experimental data over a range of physiological pH values. The NLPB equation is then used to develop an overall picture of the electrostatics of the lambdacI repressor-operator interaction. We find that long-range Coulombic forces associated with the highly charged nucleic acid provide a strong driving force for the interaction of the protein with the DNA. These favorable electrostatic interactions are opposed, however, by unfavorable changes in the solvation of both the protein and the DNA upon binding. Specifically, the formation of a protein-DNA complex removes both charged and polar groups at the binding interface from solvent while it displaces salt from around the nucleic acid. As a result, the electrostatic contribution to the lambdacI repressor-operator interaction opposes binding by approximately 73 kcal/mol at physiological salt concentrations and neutral pH. A variety of entropic terms also oppose binding. The major force driving the binding process appears to be release of interfacial water from the protein and DNA surfaces upon complexation and, possibly, enhanced packing interactions between the protein and DNA in the interface. When the various nonelectrostatic terms are described with simple models that have been applied previously to other binding processes, a general picture of protein/DNA association emerges in which binding is driven by the nonpolar interactions, whereas specificity results from electrostatic interactions that weaken binding but are necessary components of any protein/DNA complex.     

Link between protein-solvent and weak protein-protein interactions gives insight into halophilic adaptation

Malate dehydrogenase (Hm MalDH) from the extreme halophile Haloarcula marismortui is a very acidic protein with extensive ion binding properties. It is a good model for the study of solvation-solubility relationships. We measured the small-angle neutron or X-ray scattering profiles of folded and stable Hm MalDH at various protein concentrations and derived the second virial coefficients A(2). In NaCl, CsCl, KF, KCl, and NaCH(3)CO(2), A(2) values are positive, indicating globally repulsive protein-protein interactions. Below 1 M MgCl(2) and MgSO(4) or above 2 M (NH(4))(2)SO(4), A(2) rapidly decreases. From structure factor modeling with DLVO (Derjaguin, Landau, Verwey, and Overbeek)-like potentials, an effective diameter of 80-82 A is found for the protein particle in solution, compatible with its structural dimensions; the effective charge of the particle is undefined because of the high salt concentration. The strong variations of the protein-protein interaction are correlated to an attractive potential whose depth evolves with the salinity but in an opposite way in Mg salts and (NH(4))(2)SO(4). A repulsive Donnan term, corresponding to counterion dissociation, and an attractive term related to previously measured preferential salt binding parameters are discussed from well-established thermodynamics considerations and qualitatively account for the behavior of the protein-protein interactions in the various solutions. Because a solvation shell with a composition different from bulk induces protein-protein attraction, molecular adaptation to high salt would be directed to allow protein-salt interactions in order to avoid water or salt enrichment at the surface of the protein and thus preserve its solubility.     

Effect of salt concentration on the conformation of TAR RNA and its association with aminoglycoside antibiotics

RNA is an important biological target because it plays essential roles in many pathogenic and normal cellular processes. The design of inhibitors that target RNA involves optimization of noncovalent interactions, including van der Waals, hydrogen bond, and electrostatic interactions. Although sometimes regarded as nonspecific, electrostatic interactions are important in this optimization because the specific position of the phosphates may allow for specific charge-charge interactions with bound ligands. In this work, we have investigated the contribution of electrostatic interactions to the binding affinity of aminoglycoside antibiotics for TAR RNA. Because the charges in aminoglycoside antibiotics are provided by protonated amino groups, it is difficult to separate the contribution of hydrogen bonds and electrostatics to their binding specificity. Hence, we have investigated the dependence of the binding affinity on salt concentration, which should affect only the electrostatic contributions. Our results show that four aminoglycoside antibiotics (paromomycin, kanamycin-B, gentamycin, and tobramycin) bind TAR RNA with different affinities. Furthermore, the dependence of the binding affinity on salt concentration is different for kanamycin-B and paromomycin, with kanamycin-B showing a stronger dependence. Because all these antibiotics contain five positive charges, the results suggest that each antibiotic orients its charges in different ways when bound to TAR RNA. Our overall results support the idea that charge-charge interactions can contribute significantly to the specific binding of antibiotics to TAR RNA. Hence, the exact position of the charges should be considered in the design of any inhibitor of the interactions of TAR RNA.     

Role of electrostatic interactions in PDZ domain ligand recognition

PDZ domains are protein-protein interaction modules that normally recognize short C-terminal peptides. The apparent requirement for a ligand with a free terminal carboxylate group has led to the proposal that electrostatic interactions with the terminus play a significant role in recognition. However, this model has been called into question by the more recent finding that PDZ domains can recognize some internal peptide motifs that occur within a specific secondary structure context. Although these motifs bind at the same interface, they lack a terminal charge. Here we have investigated the role of electrostatics in PDZ-mediated recognition in the mouse alpha1-syntrophin PDZ domain by examining the salt dependence of binding to both terminal and internal ligands and the effects of mutating a conserved basic residue previously proposed to play a role in electrostatic recognition. These studies indicate that direct electrostatic interactions with the peptide terminus do not play a significant energetic role in binding. Additional chemical modification studies of the peptide terminus support a model in which steric and hydrogen bonding complementarity play a primary role in recognition specificity. Peptides with a free carboxy terminus, or presented within a specific structural context, can satisfy these requirements.     

Salt enhances calmodulin-target interaction

Calmodulin (CaM) operates as a Ca(2+) sensor and is known to interact with and regulate hundreds of proteins involved in a great many aspects of cellular function. It is of considerable interest to understand the balance of forces in complex formation of CaM with its target proteins. Here we have studied the importance of electrostatic interactions in the complex between CaM and a peptide derived from smooth-muscle myosin light-chain kinase by experimental methods and Monte Carlo simulations of electrostatic interactions. We show by Monte Carlo simulations that, in agreement with experimental data, the binding affinity between CaM and highly charged peptides is surprisingly insensitive to changes in the net charge of both the protein and peptide. We observe an increase in the binding affinity between oppositely charged partners with increasing salt concentration from zero to 100 mM, showing that formation of globular CaM-kinase type complexes is facilitated at physiological ionic strength. We conclude that ionic interactions in complex formation are optimized at pH and saline similar to the cell environment, which probably overrules the electrostatic repulsion between the negatively charged Ca(2+)-binding domains of CaM. We propose a conceivable rationalization of CaM electrostatics associated with interdomain repulsion.     

Nonimmunospecific protein-protein interactions of IgG: studies of the binding of IgG to IgG immunoadsorbents.

Nonimmunospecific interactions of IgG and IgG-agarose columns were systematically studied under varying conditions. Nonimmunospecific binding to the columns was primarily due to protein-protein interactions. These nonimmunospecific protein-protein interactions of IgG were enhanced with heat-induced or chemical aggregation of IgG, low pH, low ionic strength (at pH above 4), or low temperature. Conversely, this binding was decreased with proteolytic fragmentation of IgG, high ionic strength (at pH above 4), or temperatures above 4 degrees C. Chemical modification of IgG by acetylation, formalinization, carbamylation, or reaction with 1,2-cyclohexanedione significantly decreased these interactions. These observations suggest that above pH 4, ionic interactions caused the protein-protein binding. Below pH 4, hydrophobic interactions presumably play a major role. These results permit the development of rational methodology for avoiding nonimmunospecific protein-protein interactions in immunologic procedures for detection, isolation, or quantification of rheumatoid factors and other antibodies to IgG.     

Protein interactions in solution characterized by light and neutron scattering: comparison of lysozyme and chymotrypsinogen.

The effects of pH and electrolyte concentration on protein-protein interactions in lysozyme and chymotrypsinogen solutions were investigated by static light scattering (SLS) and small-angle neutron scattering (SANS). Very good agreement between the values of the virial coefficients measured by SLS and SANS was obtained without use of adjustable parameters. At low electrolyte concentration, the virial coefficients depend strongly on pH and change from positive to negative as the pH increases. All coefficients at high salt concentration are slightly negative and depend weakly on pH. For lysozyme, the coefficients always decrease with increasing electrolyte concentration. However, for chymotrypsinogen there is a cross-over point around pH 5.2, above which the virial coefficients decrease with increasing ionic strength, indicating the presence of attractive electrostatic interactions. The data are in agreement with Derjaguin-Landau-Verwey-Overbeek (DLVO)-type modeling, accounting for the repulsive and attractive electrostatic, van der Waals, and excluded volume interactions of equivalent colloid spheres. This model, however, is unable to resolve the complex short-ranged orientational interactions. The results of protein precipitation and crystallization experiments are in qualitative correlation with the patterns of the virial coefficients and demonstrate that interaction mapping could help outline new crystallization regions.     

Coarse-grained strategy for modeling protein stability in concentrated solutions

We present a coarse-grained approach for modeling the thermodynamic stability of single-domain globular proteins in concentrated aqueous solutions. Our treatment derives effective protein-protein interactions from basic structural and energetic characteristics of the native and denatured states. These characteristics, along with the intrinsic (i.e., infinite dilution) thermodynamics of folding, are calculated from elementary sequence information using a heteropolymer collapse theory. We integrate this information into Reactive Canonical Monte Carlo simulations to investigate the connections between protein sequence hydrophobicity, protein-protein interactions, protein concentration, and the thermodynamic stability of the native state. The model predicts that sequence hydrophobicity can affect how protein concentration impacts native-state stability in solution. In particular, low hydrophobicity proteins are primarily stabilized by increases in protein concentration, whereas high hydrophobicity proteins exhibit richer nonmonotonic behavior. These trends appear qualitatively consistent with the available experimental data. Although factors such as pH, salt concentration, and protein charge are also important for protein stability, our analysis suggests that some of the nontrivial experimental trends may be driven by a competition between destabilizing hydrophobic protein-protein attractions and entropic crowding effects.     

Protein-protein interactions in concentrated electrolyte solutions

Protein-protein interactions were measured for ovalbumin and for lysozyme in aqueous salt solutions. Protein-protein interactions are correlated with a proposed potential of mean force equal to the free energy to desolvate the protein surface that is made inaccessible to the solvent due to the protein-protein interaction. This energy is calculated from the surface free energy of the protein that is determined from protein-salt preferential-interaction parameter measurements. In classical salting-out behavior, the protein-salt preferential interaction is unfavorable. Because addition of salt raises the surface free energy of the protein according to the surface-tension increment of the salt, protein-protein attraction increases, leading to a reduction in solubility. When the surface chemistry of proteins is altered by binding of a specific ion, salting-in is observed when the interactions between (kosmotrope) ion-protein complexes are more repulsive than those between the uncomplexed proteins. However, salting-out is observed when interactions between (chaotrope) ion-protein complexes are more attractive than those of the uncomplexed proteins.     

Electrostatic control of calcineurin's intrinsically-disordered regulatory domain binding to calmodulin

Calcineurin (CaN) is a serine/threonine phosphatase that regulates a variety of physiological and pathophysiological processes in mammalian tissue. The calcineurin (CaN) regulatory domain (RD) is responsible for regulating the enzyme's phosphatase activity, and is believed to be highly-disordered when inhibiting CaN, but undergoes a disorder-to-order transition upon diffusion-limited binding with the regulatory protein calmodulin (CaM). The prevalence of polar and charged amino acids in the regulatory domain (RD) suggests electrostatic interactions are involved in mediating calmodulin (CaM) binding, yet the lack of atomistic-resolution data for the bound complex has stymied efforts to probe how the RD sequence controls its conformational ensemble and long-range attractions contribute to target protein binding. In the present study, we investigated via computational modeling the extent to which electrostatics and structural disorder facilitate CaM/CaN association kinetics. Specifically, we examined several RD constructs that contain the CaM binding region (CAMBR) to characterize the roles of electrostatics versus conformational diversity in controlling diffusion-limited association rates, via microsecond-scale molecular dynamics (MD) and Brownian dynamic (BD) simulations. Our results indicate that the RD amino acid composition and sequence length influence both the dynamic availability of conformations amenable to CaM binding, as well as long-range electrostatic interactions to steer association. These findings provide intriguing insight into the interplay between conformational diversity and electrostatically-driven protein-protein association involving CaN, which are likely to extend to wide-ranging diffusion-limited processes regulated by intrinsically-disordered proteins.     

Poisson-Boltzmann calculations of nonspecific salt effects on protein-protein binding free energies

The salt dependence of the binding free energy of five protein-protein hetero-dimers and two homo-dimers/tetramers was calculated from numerical solutions to the Poisson-Boltzmann equation. Overall, the agreement with experimental values is very good. In all cases except one involving the highly charged lactoglobulin homo-dimer, increasing the salt concentration is found both experimentally and theoretically to decrease the binding affinity. To clarify the source of salt effects, the salt-dependent free energy of binding is partitioned into screening terms and to self-energy terms that involve the interaction of the charge distribution of a monomer with its own ion atmosphere. In six of the seven complexes studied, screening makes the largest contribution but self-energy effects can also be significant. The calculated salt effects are found to be insensitive to force-field parameters and to the internal dielectric constant assigned to the monomers. Nonlinearities due to high charge densities, which are extremely important in the binding of proteins to negatively charged membrane surfaces and to nucleic acids, make much smaller contributions to the protein-protein complexes studied here, with the exception of highly charged lactoglobulin dimers. Our results indicate that the Poisson-Boltzmann equation captures much of the physical basis of the nonspecific salt dependence of protein-protein complexation.     

Dynamic structural rearrangements between DNA binding modes of E. coli SSB protein

Escherichia coli single-stranded (ss)DNA binding (SSB) protein binds ssDNA in multiple binding modes and regulates many DNA processes via protein-protein interactions. Here, we present direct evidence for fluctuations between the two major modes of SSB binding, (SSB)(35) and (SSB)(65) formed on (dT)(70), with rates of interconversion on time scales that vary as much as 200-fold for a mere fourfold change in NaCl concentration. Such remarkable electrostatic effects allow only one of the two modes to be significantly populated outside a narrow range of salt concentration, providing a context for precise control of SSB function in cellular processes via SSB expression levels and interactions with other proteins. Deletion of the acidic C terminus of SSB, the site of binding of several proteins involved in DNA metabolism, does not affect the strong salt dependence, but shifts the equilibrium towards the highly cooperative (SSB)(35) mode, suggesting that interactions of proteins with the C terminus may regulate the binding mode transition and vice versa. Single molecule analysis further revealed a novel low abundance binding configuration and provides a direct demonstration that the SSB-ssDNA complex is a finely tuned assembly in dynamic equilibrium among several well-defined structural and functional states.     

Preferential interactions determine protein solubility in three-component solutions: the MgCl2 system

The correlation between protein solubility and the preferential interactions of proteins with solvent components was critically examined with aqueous MgCl2 as the solvent system. Preferential interaction and solubility measurements with three proteins, beta-lactoglobulin, bovine serum albumin, and lysozyme, resulted in similar patterns of interaction. At acid pH (pH 2-3) and lower salt concentrations (less than 2 M), the proteins were preferentially hydrated, while at higher salt concentrations, the interaction was either that of preferential salt binding or low salt exclusion. At pH 4.5-5, all three proteins exhibited either very low preferential hydration or preferential binding of MgCl2. These results were analyzed in terms of the balance between salt binding and salt exclusion attributed to the increase in the surface tension of water by salts, which is invariant with conditions. It was shown that the increase in salt binding at high salt concentration is a reflection of mass action, while its decrease at acid pH is due to the electrostatic repulsion between Mg2+ ions and the high net positive charge on the protein. The preferential interaction pattern was paralleled by the variation of protein solubility with solvent conditions. Calculation of the transfer free energies from water to the salt solutions for proteins in solution and in the precipitate showed dependencies on salt concentration. This indicates that the nature of interactions between proteins and solvent components is the same in solution and in the solid state, which implies no change in protein structure during precipitation. Analysis of the transfer free energies and preferential interaction parameter in terms of the salting-in, salting-out, and weak ion binding contributions has led to the conclusions that, when the weak ion binding contribution is small, the predominant protein-salt interaction must be that of preferential salt exclusion most probably caused by the increase of the surface tension of water by addition of the salt. A necessary consequence of this is salting-out of the protein, if the protein structure is to remain unaltered.     

Influence of electrostatic forces on the association kinetics and conformational ensemble of an intrinsically disordered protein

Recent work has revealed that the association of a disordered region of a protein with a folded binding partner can occur as rapidly as association between two folded proteins. This is the case for the phosphatase calcineurin (CaN) and its association with its activator calmodulin. Calmodulin binds to the intrinsically disordered regulatory domain of CaN. Previous studies have shown that electrostatic steering can accelerate the binding of folded proteins with disordered ligands. Given that electrostatic forces are strong determinants of disordered protein ensembles, the relationship between electrostatics, conformational ensembles, and quaternary interactions is unclear. Here, we employ experimental approaches to explore the impact of electrostatic interactions on the association of calmodulin with the disordered regulatory region of CaN. We find that estimated association rate constants of calmodulin with our chosen calmodulin-substrates are within the diffusion-limited regime. The association rates are dependent on the ionic strength, indicating that favorable electrostatic forces increase the rate of association. Further, we show that charged amino acids outside the calmodulin-binding site modulate the binding rate. Conformational ensembles obtained from computer simulations suggest that electrostatic interactions within the regulatory domain might bias the conformational ensemble such that the calmodulin binding region is readily accessible. Given the prevalence of charged residues in disordered protein chains, our findings are likely relevant to many protein-protein interactions.