SCORE: A New Empirical Method for Estimating the Binding Affinity of a Protein-Ligand Complex

A new method is presented to estimate the binding affinity of a protein-ligand complex with known three-dimensional structure. The method, SCORE, uses an empirical scoring function to describe the binding free energy, which includes terms to account for van der Waals contact, metal-ligand bonding, hydrogen bonding, desolvation effect, and deformation penalty upon the binding process. The coefficients of each term are obtained by multivariate regressional analysis of a diverse training set of 170 protein-ligand complexes. The final scoring function reproduces the binding free energies of the whole training set with a cross-validated deviation of 6.3 kJ/mol. The predictive ability of the function is further tested by a set of 11 endothiapepsin complexes and the internal consistency of the function is demonstrated in a stepwise procedure named Evolutionary Test. A major innovation of this method is the introduction of an atomic binding score which allows the researcher to inspect and optimize the lead compound rationally in a structure-based drug design scheme.     

The Affinity of DNA-Microtubule Protein Complexes and their Disruption by Tubulin Binding Drugs

Abstract

Using the gel shift assay system, we have measured the apparent affinity constant for the interaction of two different DNAs with MAP proteins found in both total calf brain microtubules and heat stable brain preparations. Both DNAs studied contained centromere/kinetochore sequences- one was enriched in the calf satellite DNA; the other was a large restriction fragment containing the yeast CEN11 DNA sequence. Complexes formed using both DNAs had similar K_app values in the range of 2.1×10⁷ M^?1 to 2.0×10⁸ M^?1. CEN11 DNA-MTP complexes had by far the highest K_app value of 2.0×10⁸ M^?1. The CEN11 DNA sequence is where the yeast kinetochore of chromosome 11 is formed and where the single yeast microtubule is bound in vivo. The CEN11 conserved region II known binding sites -(dA/dT)n runs- for mammalian MAP2 protein, are in good agreement with this higher K_app value. The effects of the classical tubulin binding drugs colchicine, podophyllotoxin and vinblastine on the DNA-MAP protein complex stability were investigated by determining the drug concentrations where the complexes were destabilized. Only the complexes formed from total microtubule protein (tubulin containing) were destabilized over a wide drug concentration range. Heat stable brain protein complexes (no tubulin) were largely unaffected. Furthermore, it took 10–100 fold higher drug concentrations to disrupt the CEN 11 DNA complexes compared to the calf thymus satellite DNA enriched complexes. These data support our previous results suggesting that there is a DNA sequence dependent interaction with MAP proteins that appears to be conserved in evolution (Marx et. al., Biochim. Biophys. Acta. 783, 383–392,1984; Marx and Denial, Molecular Basis of Cancer 172B,65-15 1985). In addition, these results imply that the classical tubulin binding drugs may exert their biological effects in cells at least in part by disrupting DNA-Protein complexes of the type we have studied here.     

Entropic Enhancement of Protein-DNA Affinity by Oxygen-to-Sulfur Substitution in DNA Phosphate

Dithioation of DNA phosphate is known to enhance binding affinities, at least for some proteins. We mechanistically characterized this phenomenon for the Antennapedia homeodomain-DNA complex by integrated use of fluorescence, isothermal titration calorimetry, NMR spectroscopy, and x-ray crystallography. By fluorescence and isothermal titration calorimetry, we found that this affinity enhancement is entropy driven. By NMR, we investigated the ionic hydrogen bonds and internal motions of lysine side-chain NH₃⁺ groups involved in ion pairs with DNA. By x-ray crystallography, we compared the structures of the complexes with and without dithioation of the phosphate. Our NMR and x-ray data show that the lysine side chain in contact with the DNA phosphate becomes more dynamic upon dithioation. Our thermodynamic, structural, and dynamic investigations collectively suggest that the affinity enhancement by the oxygen-to-sulfur substitution in DNA phosphate is largely due to an entropic gain arising from mobilization of the intermolecular ion pair at the protein-DNA interface.     

Determining the Binding Affinity of Therapeutic Monoclonal Antibodies towards Their Native Unpurified Antigens in Human Serum

Monoclonal antibodies (mAbs) are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen’s endogenous concentration, by combining the kinetic exclusion assay and Biacore’s calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9), progranulin (PGRN), and fatty acid binding protein (FABP4). We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.     

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh

Glt_Ph from Pyrococcus horikoshii is a homotrimeric Na⁺-coupled aspartate transporter. It belongs to the widespread family of glutamate transporters, which also includes the mammalian excitatory amino acid transporters that take up the neurotransmitter glutamate. Each protomer in Glt_Ph consists of a trimerization domain involved in subunit interactions and a transport domain containing the substrate binding site. Here, we have studied the dynamics of Na⁺ and aspartate binding to Glt_Ph. Tryptophan fluorescence measurements on the fully active single tryptophan mutant F273W revealed that Na⁺ binds with low affinity to the apoprotein (K_d 120 mm), with a particularly low k_on value (5.1 m⁻¹s⁻¹). At least two sodium ions bind before aspartate. The binding of Na⁺ requires a very high activation energy (E_a 106.8 kJ mol⁻¹) and consequently has a large Q₁₀ value of 4.5, indicative of substantial conformational changes before or after the initial binding event. The apparent affinity for aspartate binding depended on the Na⁺ concentration present. Binding of aspartate was not observed in the absence of Na⁺, whereas in the presence of high Na⁺ concentrations (above the K_d for Na⁺) the dissociation constants for aspartate were in the nanomolar range, and the aspartate binding was fast (k_on of 1.4 × 10⁵ m⁻¹s⁻¹), with low E_a and Q₁₀ values (42.6 kJ mol⁻¹ and 1.8, respectively). We conclude that Na⁺ binding is most likely the rate-limiting step for substrate binding.     

Statistics and Physical Origins of pK and Ionization State Changes upon Protein-Ligand Binding

This work investigates statistical prevalence and overall physical origins of changes in charge states of receptor proteins upon ligand binding. These changes are explored as a function of the ligand type (small molecule, protein, and nucleic acid), and distance from the binding region. Standard continuum solvent methodology is used to compute, on an equal footing, pK changes upon ligand binding for a total of 5899 ionizable residues in 20 protein-protein, 20 protein-small molecule, and 20 protein-nucleic acid high-resolution complexes. The size of the data set combined with an extensive error and sensitivity analysis allows us to make statistically justified and conservative conclusions: in 60% of all protein-small molecule, 90% of all protein-protein, and 85% of all protein-nucleic acid complexes there exists at least one ionizable residue that changes its charge state upon ligand binding at physiological conditions (pH = 6.5). Considering the most biologically relevant pH range of 4-8, the number of ionizable residues that experience substantial pK changes (ΔpK > 1.0) due to ligand binding is appreciable: on average, 6% of all ionizable residues in protein-small molecule complexes, 9% in protein-protein, and 12% in protein-nucleic acid complexes experience a substantial pK change upon ligand binding. These changes are safely above the statistical false-positive noise level. Most of the changes occur in the immediate binding interface region, where approximately one out of five ionizable residues experiences substantial pK change regardless of the ligand type. However, the physical origins of the change differ between the types: in protein-nucleic acid complexes, the pK values of interface residues are predominantly affected by electrostatic effects, whereas in protein-protein and protein-small molecule complexes, structural changes due to the induced-fit effect play an equally important role. In protein-protein and protein-nucleic acid complexes, there is a statistically significant number of substantial pK perturbations, mostly due to the induced-fit structural changes, in regions far from the binding interface.     

AutoDockFR: Advances in Protein-Ligand Docking with Explicitly Specified Binding Site Flexibility

Automated docking of drug-like molecules into receptors is an essential tool in structure-based drug design. While modeling receptor flexibility is important for correctly predicting ligand binding, it still remains challenging. This work focuses on an approach in which receptor flexibility is modeled by explicitly specifying a set of receptor side-chains a-priori. The challenges of this approach include the: 1) exponential growth of the search space, demanding more efficient search methods; and 2) increased number of false positives, calling for scoring functions tailored for flexible receptor docking. We present AutoDockFR–AutoDock for Flexible Receptors (ADFR), a new docking engine based on the AutoDock4 scoring function, which addresses the aforementioned challenges with a new Genetic Algorithm (GA) and customized scoring function. We validate ADFR using the Astex Diverse Set, demonstrating an increase in efficiency and reliability of its GA over the one implemented in AutoDock4. We demonstrate greatly increased success rates when cross-docking ligands into apo receptors that require side-chain conformational changes for ligand binding. These cross-docking experiments are based on two datasets: 1) SEQ17 –a receptor diversity set containing 17 pairs of apo-holo structures; and 2) CDK2 –a ligand diversity set composed of one CDK2 apo structure and 52 known bound inhibitors. We show that, when cross-docking ligands into the apo conformation of the receptors with up to 14 flexible side-chains, ADFR reports more correctly cross-docked ligands than AutoDock Vina on both datasets with solutions found for 70.6% vs. 35.3% systems on SEQ17, and 76.9% vs. 61.5% on CDK2. ADFR also outperforms AutoDock Vina in number of top ranking solutions on both datasets. Furthermore, we show that correctly docked CDK2 complexes re-create on average 79.8% of all pairwise atomic interactions between the ligand and moving receptor atoms in the holo complexes. Finally, we show that down-weighting the receptor internal energy improves the ranking of correctly docked poses and that runtime for AutoDockFR scales linearly when side-chain flexibility is added.     

Ion Channel Selectivity through Stepwise Changes in Binding Affinity

Voltage-gated Ca²⁺ channels select Ca²⁺ over competing, more abundant ions by means of a high affinity binding site in the pore. The maximum off rate from this site is ∼1,000× slower than observed Ca²⁺ current. Various theories that explain how high Ca²⁺ current can pass through such a sticky pore all assume that flux occurs from a condition in which the pore''s affinity for Ca²⁺ transiently decreases because of ion interactions. Here, we use rate theory calculations to demonstrate a different mechanism that requires no transient changes in affinity to quantitatively reproduce observed Ca²⁺ channel behavior. The model pore has a single high affinity Ca²⁺ binding site flanked by a low affinity site on either side; ions permeate in single file without repulsive interactions. The low affinity sites provide steps of potential energy that speed the exit of a Ca²⁺ ion off the selectivity site, just as potential energy steps accelerate other chemical reactions. The steps could be provided by weak binding in the nonselective vestibules that appear to be a general feature of ion channels, by specific protein structures in a long pore, or by stepwise rehydration of a permeating ion. The previous ion-interaction models and this stepwise permeation model demonstrate two general mechanisms, which might well work together, to simultaneously generate high flux and high selectivity in single file pores.     

Non-additivity of Functional Group Contributions in Protein-Ligand Binding: A Comprehensive Study by Crystallography and Isothermal Titration Calorimetry

Additivity of functional group contributions to protein-ligand binding is a very popular concept in medicinal chemistry as the basis of rational design and optimized lead structures. Most of the currently applied scoring functions for docking build on such additivity models. Even though the limitation of this concept is well known, case studies examining in detail why additivity fails at the molecular level are still very scarce. The present study shows, by use of crystal structure analysis and isothermal titration calorimetry for a congeneric series of thrombin inhibitors, that extensive cooperative effects between hydrophobic contacts and hydrogen bond formation are intimately coupled via dynamic properties of the formed complexes. The formation of optimal lipophilic contacts with the surface of the thrombin S3 pocket and the full desolvation of this pocket can conflict with the formation of an optimal hydrogen bond between ligand and protein. The mutual contributions of the competing interactions depend on the size of the ligand hydrophobic substituent and influence the residual mobility of ligand portions at the binding site. Analysis of the individual crystal structures and factorizing the free energy into enthalpy and entropy demonstrates that binding affinity of the ligands results from a mixture of enthalpic contributions from hydrogen bonding and hydrophobic contacts, and entropic considerations involving an increasing loss of residual mobility of the bound ligands. This complex picture of mutually competing and partially compensating enthalpic and entropic effects determines the non-additivity of free energy contributions to ligand binding at the molecular level.     

Binding Specificity and Affinity of Acriflavine for Nucleic Acids

A set of conditions was developed for the specific binding of acriflavine to the DNA of intact squamous cells. This was achieved through a series of studies into the relative affinities for dye between DNA and various biopolymers by an agar gel diffusion technique. Specificity was ascertained by DNase and RNase treatment of the cells. The final conditions, based on an estimated DNA-to-dye ratio of 4:1, required a constant cell count of 100,000 and dye at a concentration of 0.0025 μg per ml in 10 ml of phosphate buffer, pH 6.0-7.4. These quantities were dictated by the sensitivity limitations of the analytical apparatus. To make use of standard fluorometric instrumentation, the whole cell population method for determining average values was followed. Free dye was analyzed after cell samples were stained, and the difference between this value and that of an aliquot of working dye was taken as the amount of bound dye. To ensure cell-free residues of dye, cells were removed by centrifugation through Teflon membrane filters. The average amount of dye which was bound to the DNA of normal squamous cells was 1.6 × 10^-7 μg (0.7 × 10^-15 mole) per cell, and the amount bound to HeLa cells was 2.3 × 10^-7 μg (1 × 10^-15 mole). These values were highly replicable, making it possible to use them as an expression of the DNA content of the cells. The mildness of the staining conditions, and the preservation of cellular integrity in this technique may permit quantitative measurements of the DNA content of living cells.     

High Affinity Binding of Indium and Ruthenium Ions by Gastrins

The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga³⁺ were fitted by a 2 site model with dissociation constants (K_d) of 3.3 x 10⁻⁷ and 1.1 x 10⁻⁶ M. Although the absorption of gastrin did not change upon the addition of In³⁺ ions, the changes in absorbance on Fe³⁺ ion binding in the presence of indium ions were fitted by a 2 site model with K_d values for In³⁺ of 6.5 x 10⁻¹⁵ and 1.7 x 10⁻⁷ M. Similar results were obtained with Ru³⁺ ions, although the K_d values for Ru³⁺ of 2.6 x 10⁻¹³ and 1.2 x 10⁻⁵ M were slightly larger than observed for In³⁺. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0–3.3 Å, the Ru complex clearly demonstrated a short range Ru—Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In³⁺ or Ru³⁺ ions, and that the affinity of the first site for In³⁺ or Ru³⁺ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.     

Fusion of Carbohydrate Binding Modules to Bifunctional Cellulase to Enhance Binding Affinity and Cellulolytic Activity

Bifunctional cellulase (glycoside hydrolase 5, GH5) from Bacillus sp. D04 having both endo- and exoglucanase activities was fused with two types of carbohydrate binding modules (CBMs). CBM3 from Bacillus sp. D04 and CBM9 from Thermotoga maritima Xyn10A were added to GH5 to hydrolyze microcrystalline cellulose (Avicel) as well as water-soluble cellulose (carboxymethyl cellulose, CMC). The optimum temperature of GH5 was 50oC, while it increased to 60oC for the fusion GH5-CBM3 and GH5-CBM9, indicating that addition of CBM increased the thermostability of the enzyme. Addition of CBM3 and CBM9 enhanced the GH5 affinity (K_M), for which K_M decreased from 104 to 33.9 ~ 35.1 mg/mL for CMC, and from 115 to 55.5 ~ 80.3 mg/mL for Avicel, respectively. The catalytic efficiency (k_cat/K_M) also increased from 4.80 to 5.36 ~ 6.46 (mL/mg)/sec for CMC, and from 1.77 to 2.40 ~ 4.45 (mL/mg)/sec for Avicel, respectively, by addition of CBM3 and CBM9.     

Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.     

Predicting Ligand Binding Affinity with Alchemical Free Energy Methods in a Polar Model Binding Site

We present a combined experimental and modeling study of organic ligand molecules binding to a slightly polar engineered cavity site in T4 lysozyme (L99A/M102Q). For modeling, we computed alchemical absolute binding free energies. These were blind tests performed prospectively on 13 diverse, previously untested candidate ligand molecules. We predicted that eight compounds would bind to the cavity and five would not; 11 of 13 predictions were correct at this level. The RMS error to the measurable absolute binding energies was 1.8 kcal/mol. In addition, we computed “relative” binding free energies for six phenol derivatives starting from two known ligands: phenol and catechol. The average RMS error in the relative free energy prediction was 2.5 kcal/mol (phenol) and 1.1 kcal/mol (catechol). To understand these results at atomic resolution, we obtained x-ray co-complex structures for nine of the diverse ligands and for all six phenol analogs. The average RMSD of the predicted pose to the experiment was 2.0 Å (diverse set), 1.8 Å (phenol-derived predictions), and 1.2 Å (catechol-derived predictions). We found that predicting accurate affinities and rank-orderings required near-native starting orientations of the ligand in the binding site. Unanticipated binding modes, multiple ligand binding, and protein conformational change all proved challenging for the free energy methods. We believe that these results can help guide future improvements in physics-based absolute binding free energy methods.     

ATP Binding and Hydrolysis by Mcm2 Regulate DNA Binding by Mcm Complexes

The essential minichromosome maintenance (Mcm) proteins Mcm2 through Mcm7 likely comprise the replicative helicase in eukaryotes. In addition to Mcm2-7, other subcomplexes, including one comprising Mcm4, Mcm6, and Mcm7, unwind DNA. Using Mcm4/6/7 as a tool, we reveal a role for nucleotide binding by Saccharomyces cerevisiae Mcm2 in modulating DNA binding by Mcm complexes. Previous studies have shown that Mcm2 inhibits DNA unwinding by Mcm4/6/7. Here, we show that interaction of Mcm2 and Mcm4/6/7 is not sufficient for inhibition; rather, Mcm2 requires nucleotides for its regulatory role. An Mcm2 mutant that is defective for ATP hydrolysis (K549A), as well as ATP analogues, was used to show that ADP binding by Mcm2 is required to inhibit DNA binding and unwinding by Mcm4/6/7. This Mcm2-mediated regulation of Mcm4/6/7 is independent of Mcm3/5. Furthermore, the importance of ATP hydrolysis by Mcm2 to the regulation of the native complex was apparent from the altered DNA binding properties of Mcm2^KA-7. Moreover, together with the finding that Mcm2_K549A does not support yeast viability, these results indicate that the nucleotide-bound state of Mcm2 is critical in regulating the activities of Mcm4/6/7 and Mcm2-7 complexes.