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.     

Potential of mean force for protein-protein interaction studies.

Calculating protein-protein interaction energies is crucial for understanding protein-protein associations. On the basis of the methodology of mean-field potential, we have developed an empirical approach to estimate binding free energy for protein-protein interactions. This knowledge-based approach has been used to derive distance-dependent free energies of protein complexes from a nonredundant training set in the Protein Data Bank (PDB), with a careful treatment of homology. We calculate atom pair potentials for 16 pair interactions, which can reflect the importance of hydrophobic interactions and specific hydrogen-bonding interactions. The derived potentials for hydrogen-bonding interactions show a valley of favorable interactions at a distance of approximately 3 A, corresponding to that of an established hydrogen bond. For the test set of 28 protein complexes, the calculated energies have a correlation coefficient of 0.75 compared with experimental binding free energies. The performance of the method in ranking the binding energies of different protein-protein complexes shows that the energy estimation can be applied to value binding free energies for protein-protein associations.     

Selection of an improved HDAC8 inhibitor through structure-based drug design

Histone deacetylases (HDACs) are enzymes, which catalyze the removal of acetyl moiety from acetyl-lysine within the histone proteins and promote gene repression and silencing resulting in several types of cancer. HDACs are important therapeutic targets for the treatment of cancer and related diseases. Hydroxamic acid inhibitors show promising results in clinical trials against carcinogenesis. 120 hydroxamic acid derivatives were designed as inhibitors based on hydrophobic pocket and the Zn (II) catalytic site of HDAC8 active site using Structure Based Drug Design (SBDD) approach. High Throughput Virtual screening (HTVs) was used to filter the effective inhibitors. Induced Fit Docking (IFD) studies were carried out for the screening of eight inhibitors using Glide software. Hydrogen bond, hydrophobic interactions and octahedral coordination geometry with Zn (II) were observed in the IFD complexes. Prime MM-GBSA calculation was carried out for the binding free energy, to observe the stability of docked complexes. The Lipinski's rule of five was analyzed for ADME/Tox drug likeliness using Qikprop simulation. These inhibitors have good inhibitory properties as they have favorable docking score, energy, emodel, hydrogen bond and hydrophobic interactions, binding free energy and ADME/Tox. However, one compound (Cmp22) successively satisfied all the studies among the eight compounds screened and seems to be a promising potent inhibitor against HDAC8.     

Thermodynamic cycle analysis and inhibitor design against beta-lactamase

Beta-lactamases are the most widespread resistance mechanism to beta-lactam antibiotics, such as the penicillins and cephalosporins. Transition-state analogues that bind to the enzymes with nanomolar affinities have been introduced in an effort to reverse the resistance conferred by these enzymes. To understand the origins of this affinity, and to guide design of future inhibitors, double-mutant thermodynamic cycle experiments were undertaken. An unexpected hydrogen bond between the nonconserved Asn289 and a key inhibitor carboxylate was observed in the X-ray crystal structure of a 1 nM inhibitor (compound 1) in complex with AmpC beta-lactamase. To investigate the energy of this hydrogen bond, the mutant enzyme N289A was made, as was an analogue of 1 that lacked the carboxylate (compound 2). The differential affinity of the four different protein and analogue complexes indicates that the carboxylate-amide hydrogen bond contributes 1.7 kcal/mol to overall binding affinity. Synthesis of an analogue of 1 where the carboxylate was replaced with an aldehyde led to an inhibitor that lost all this hydrogen bond energy, consistent with the importance of the ionic nature of this hydrogen bond. To investigate the structural bases of these energies, X-ray crystal structures of N289A/1 and N289A/2 were determined to 1.49 and 1.39 A, respectively. These structures suggest that no significant rearrangement occurs in the mutant versus the wild-type complexes with both compounds. The mutant enzymes L119A and L293A were made to investigate the interaction between a phenyl ring in 1 and these residues. Whereas deletion of the phenyl itself diminishes affinity by 5-fold, the double-mutant cycles suggest that this energy does not come through interaction with the leucines, despite the close contact in the structure. The energies of these interactions provide key information for the design of improved inhibitors against beta-lactamases. The high magnitude of the ion-dipole interaction between Asn289 and the carboxylate of 1 is consistent with the idea that ionic interactions can provide significant net affinity in inhibitor complexes.     

Raman spectroscopic studies of NAD coenzymes bound to malate dehydrogenases by difference techniques

We report here on the Raman spectra of NADH, 3-acetylpyridine adenine dinucleotide, APAD+, and a fragment of these molecules, adenosine 5'-diphosphate ribose (ADPR) bound to the mitochondrial (mMDH) and cytoplasmic (or soluble, sMDH) forms of malate dehydrogenase. We observe changes in the Raman spectrum of the adenosine moiety of these cofactors upon binding to mMDH, indicating that the binding site is hydrophobic. On the other hand, there is little change in the spectrum of the adenosine moiety when it binds to sMDH. Such observations are in clear contrast with those results obtained in LDH and LADH, where there are significant changes in the spectrum of the adenosine moiety when it binds to these two proteins. A strong hydrogen bond is postulated to exist between amide carbonyl group of NAD+ and the enzyme in the binary complexes with both mMDH and sMDH on the basis of a sizable decrease in the frequency of the carbonyl double bond. The interaction energy for formation of a hydrogen bond is the same as found previously for LDH, and we estimate that it is 2.8 kcal/mol more favorable in the binary complex than in water. A hydrogen bond is also detected between the amide-NH2 group of NADH and sMDH that is stronger than that formed in water and is of the same size as found in LDH. Surprisingly, the hydrogen bond to the -NH2 group in mMDH is the same as that found for water.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of water mediating protein-ligand interactions in cytochrome P450cam: a molecular dynamics study.

Ordered water molecules are observed by crystallography and nuclear magnetic resonance to mediate protein-ligand interactions. Here, we examine the energetics of hydrating cavities formed at protein-ligand interfaces using molecular dynamics simulations. The free energies of hydrating two cavities in the active site of two liganded complexes of cytochrome P450cam were calculated by multiconfigurational thermodynamic integration. The complex of cytochrome P450cam with 2-phenyl-imidazole contains a crystallographically well defined water molecule mediating hydrogen bonds between the protein and the inhibitor. We calculate that this water molecule is stabilized by a binding free energy of -11.6 +/- kJ/mol. The complex of cytochrome P450cam with its natural substrate, camphor, contains a cavity that is empty in the crystal structure although a water molecule in it could make a hydrogen bond to camphor. Here, solvation of this cavity is calculated to be unfavorable by +15.8 +/- 5.0 kJ/mol. The molecular dynamics simulations can thus distinguish a hydrated interfacial cavity from an empty one. They also provide support for the notion that protein-ligand complexes can accommodate empty interfacial cavities and that such cavities are likely to be unhydrated unless more than one hydrogen bond can be made to a water molecule in the cavity.     

Energetics of the protein-DNA-water interaction

Background  

To understand the energetics of the interaction between protein and DNA we analyzed 39 crystallographically characterized complexes with the HINT (Hydropathic INTeractions) computational model. HINT is an empirical free energy force field based on solvent partitioning of small molecules between water and 1-octanol. Our previous studies on protein-ligand complexes demonstrated that free energy predictions were significantly improved by taking into account the energetic contribution of water molecules that form at least one hydrogen bond with each interacting species.     

Vibrational normal modes and dynamical stability of DNA triplex poly(dA). 2poly(dT): S-type structure is more stable and in better agreement with observations in solution.

A normal-mode and statistical mechanical calculation was carried out to determine the vibrational normal modes, contribution of internal fluctuations to the free energy, and hydrogen bond disruption of DNA triplex poly(dA).2poly(dT). The calculation was performed on both the x-ray fiber diffraction model with a N-type sugar conformation, and a newly proposed model with a S-type sugar conformation. Our calculated normal modes for the S-type structure are in better agreement with observed IR spectra for samples in D2O solution. We also find that the contribution of internal fluctuations to free energy, premelting hydrogen bond disruption probability, and hydrogen bond melting temperatures for the Hoogsteen and Watson-Crick hydrogen bonds all show that the S-type structure is dynamically more stable than the N-type structure in a nominal solution environment. Therefore our calculation supports experimental findings that the triplex d(T)n.d(A)nd(T)n most likely adopts a S-type sugar conformation in solution or at high humidity. Our calculations, however, do not preclude the possibility of an N-type conformation at lower humidities.     

Role of Hydrogen Bonds in Protein-DNA Recognition: A Comparison of Generalized Born and Finite Difference Poisson-Boltzmann Solvation Treatments

Abstract

Hydrogen bonds have been accredited with a major role historically, in the formation and stabilization of biomolecular structures. The formation of hydrogen bonds at protein-DNA interfaces in aqueous medium involves not only favorable interactions of the donor and acceptor functional groups but also a loss of interactions between these groups with the solvent water. We have investigated the energetics of about 500 potential hydrogen bonds occuring at protein-DNA interfaces incorporating some recent improvements in biomolecular force fields and solvation treatments. We present here results of our assessment of hydrogen bond contributions to the overall standard free energy of formation of protein-DNA complexes obtained with the generalized Born model and finite difference Poisson- Boltzmann methodology for solvation in conjunction with AMBER force field. Our results support the emerging view on the role of electrostatics in general and that of hydrogen bonds in particular which is that hydrogen bonds do not drive protein-DNA complex formation by virtue of the unfavourable cost of the electrostatics of desolvation. They however, act to stabilize the complex once it is formed.     

Analysis of the heat capacity dependence of protein folding.

This paper presents an analysis of plots of enthalpy versus heat capacity change at 25 degrees C for the unfolding of proteins and for the dissolution of gaseous, liquid and solid solutes, first reported by Murphy, Privalov & Gill. The negative slope in the enthalpy plot for proteins is interpreted as arising from a large penalty associated with burying polar groups in the protein interior. The small enthalpy changes that accompany protein unfolding at 25 degrees C are also discussed. It is argued that the combined effects of hydrogen bond formation and close packing predict a large positive enthalpy of unfolding. Electrostatic calculations indicate that the penalty associated with burying polar groups is large enough to effectively cancel these terms, leading to the small net enthalpy changes that are observed. The free energy changes associated with protein folding are also discussed. The free energy cost of burying polar groups largely compensates for the stabilizing contribution of the hydrophobic effect and would appear to account for the fact that proteins are marginally stable, independent of their size and of their relative hydrophobicities.     

Aromatic rings act as hydrogen bond acceptors

Simple energy calculations show that there is a significant interaction between a hydrogen bond donor (like the greater than NH group) and the centre of a benzene ring, which acts as a hydrogen bond acceptor. This interaction, which is about half as strong as a normal hydrogen bond, contributes approximately 3 kcal/mol (1 cal = 4.184 J) of stabilizing enthalpy and is expected to play a significant role in molecular associations. It is of interest that the aromatic hydrogen bond arises from small partial charges centred on the ring carbon and hydrogen atoms: there is no need to consider delocalized electrons. Although some energy calculations have included such partial charges, their role in forming such a strong interaction was not appreciated until after aromatic hydrogen bonds had been observed in protein-drug complexes.     

Hydration of a hydrophobic cavity and its functional role: a simulation study of human interleukin-1beta

Molecular dynamics simulations reveal that the hydrophobic cavity in human cytokine Interleukin-1beta is hydrated and can dynamically accommodate between one and four water molecules. These waters have residence times > 500 ps and can give rise to detectable NOEs, in agreement with NMR observations of Ernst et al. (Science 1995; 267:1813-1817). The waters also display high positional disorder within the cavity, which explains why they have not been resolved crystallographically. The average distribution of water molecules over time within the cavity matches well the low resolution electron density extracted by Yu et al. (Proc Natl Acad Sci 1999; 96:103-108). The water molecules hydrate the hydrophobic cavity preferentially as complex clusters. These clusters result from a combination of hydrogen bonds between the waters and stabilizing interactions between the waters and aromatic rings forming the cavity. Free energy estimates suggest that it takes 4-waters to hydrate the cavity in a thermodynamically stable manner leading to a gain in free energy of transfer from bulk of approximately approximately 3.6 kcal/mol. This arises from the existence of the water clusters in multiple hydrogen bonded states. In addition, the waters are found to migrate either individually or as clusters out of the cavity through several pathways. The upper limit for one-dimensional diffusion of the waters within the protein matrix is 4 A/ps (relative to 6 A/ps for bulk). Simulations reveal pathways in addition to those identified crystallographically, with motions controlled by the rotations of sidechains. We find that only when the hydrophobic cavity is hydrated, do correlated motions couple distant sites with the sites that make contact with the receptor and this data partly offers an explanation of experimental mutagenesis data. Simulations, together with recent observations based on mutagenesis by Heidary et al. (J Mol Biol 2005; 353:1187-1198) that hydrogen bond networks couple motions across long distances in interleukin-1beta, lead us to hypothesize that the hydration of the cavity (conserved across mammals) can thermodynamically enhance hydrogen bond networks to enable coupling across long distances by acting as a plug and this in turn enables a kinetic control of the rate of transmission of signals.     

The effect of anesthetics on hydrogen bonds An infrared study at low anesthetic concentrations

It is shown that a striking parallelism exists between the anesthetic potency of general halocarbon anesthetics and their influence on the hydrogen bond association constants in N-H…OC type hydrogen bonds, important for shaping the ion channels. It is further shown that the effect of potent anesthetics (which contain an acidic hydrogen) on the free/associated ratio in such hydrogen bonds is still significant at clinical anesthetic concentrations. It is argued that the results are in keeping with a pluralistic theory of anesthesia based on both hydrophobic and polar interactions.     

Density functional theory investigation of cocaine water complexes

Twenty cocaine–water complexes were studied using density functional theory (DFT) B3LYP/6-311++G** level to understand their geometries, energies, vibrational frequencies, charge transfer and topological parameters. Among the 20 complexes, 12 are neutral and eight are protonated in the cocaine-water complexes. Based on the interaction energy, the protonated complexes are more stable than the neutral complexes. In both complexes, the most stable structure involves the hydrogen bond with water at nitrogen atom in the tropane ring and C?=?O groups in methyl ester. Carbonyl groups in benzoyl and methyl ester is the most reactive site in both forms and it is responsible for the stability order. The calculated topological results show that the interactions involved in the hydrogen bond are electrostatic dominant. Natural bond orbital (NBO) analysis confirms the presence of hydrogen bond and it supports the stability order. Atoms in molecules (AIM) and NBO analysis confirms the C-H?·?·?·?O hydrogen bonds formed between the cocaine-water complexes are blue shifted in nature.     

Energy of hydrogen bonds probed by the adhesion of functionalized lipid layers

It is now well admitted that hydrophobic interactions and hydrogen bonds are the main forces driving protein folding and stability. However, because of the complex structure of a protein, it is still difficult to separate the different energetic contributions and have a reliable estimate of the hydrogen bond part. This energy can be quantified on simpler systems such as surfaces bearing hydrogen-bonding groups. Using the surface force apparatus, we have directly measured the interaction energy between monolayers of lipids whose headgroups can establish hydrogen bonds in water: nitrilotriacetate, adenosine, thymidine, and methylated thymidine lipids. From the adhesion energy between the surfaces, we have deduced the energy of a single hydrogen bond in water. We found in each case an energy of 0.5 kcal/mol. This result is in good agreement with recent experimental and theoretical studies made on protein systems showing that intramolecular hydrogen bonds make a positive contribution to protein stabilization.     

Hydrogen bond connectivity patterns and hydrophobic interactions in crystal structures of small, acyclic peptides.

Crystal structures of all available unblocked linear peptides with two to five residues were retrieved from the Cambridge Structural Database and their intermolecular contacts and packing modes studied using molecular graphics. This survey reveals that interactions between hydrophobic portions of the molecules are critically important in determining the overall features of their crystal packing patterns. Distinct hydrophobic columns or layers are observed in almost all crystal structures. Analyses of the relationships between these interactions and crystal growth properties of small peptides are given. It is suggested that needle growth is promoted by hydrophobic packing, usually along a short crystallographic axis (4.6-6.0 angstroms). Also contributing to these morphologic characteristics are entropic factors associated with hydrophobic aggregation as well as tightly bound water molecules on hydrophobic faces. The paper also provides a comprehensive overview of hydrogen bond patterns in acyclic peptide crystals. It is demonstrated that one of their primary roles is to provide a scaffolding within which hydrophobic groups can aggregate. Even though there is a high density of hydrogen bonds in the crystals, often with complex patterns and networks, certain motifs are found to recur in a number of structures indicating specific hydrogen bond preferences. Water, for example, is an integral part of the hydrogen bond networks in these crystals, usually acting as the primary donor for main-chain carboxylate groups in peptide hydrates.     

Antifreeze proteins at the ice/water interface: three calculated discriminating properties for orientation of type I proteins

Antifreeze proteins (AFPs) protect many plants and organisms from freezing in low temperatures. Of the different AFPs, the most studied AFP Type I from winter flounder is used in the current computational studies to gain molecular insight into its adsorption at the ice/water interface. Employing molecular dynamics simulations, we calculate the free energy difference between the hydrophilic and hydrophobic faces of the protein interacting with ice. Furthermore, we identify three properties of Type I "antifreeze" proteins that discriminate among these two orientations of the protein at the ice/water interface. The three properties are: the "surface area" of the protein; a measure of the interaction of the protein with neighboring water molecules as determined by the number of hydrogen bond count, for example; and the side-chain orientation angles of the threonine residues. All three discriminants are consistent with our free energy results, which clearly show that the hydrophilic protein face orientations toward the ice/water interface, as hypothesized from experimental and ice/vacuum simulations, are incorrect and support the hypothesis that the hydrophobic face is oriented toward the ice/water interface. The adsorption free energy is calculated to be 2-3 kJ/mol.     

Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes.

In order to obtain a better structural framework for understanding the catalytic mechanism of carbonic anhydrase, a number of inhibitor complexes of the enzyme were investigated crystallographically. The three-dimensional structure of free human carbonic anhydrase II was refined at pH 7.8 (1.54 A resolution) and at pH 6.0 (1.67 A resolution). The structure around the zinc ion was identical at both pH values. The structure of the zinc-free enzyme was virtually identical with that of the native enzyme, apart from a water molecule that had moved 0.9 A to fill the space that would be occupied by the zinc ion. The complexes with the anionic inhibitors bisulfite and formate were also studied at neutral pH. Bisulfite binds with one of its oxygen atoms, presumably protonized, to the zinc ion and replaces the zinc water. Formate, lacking a hydroxyl group, is bound with its oxygen atoms not far away from the position of the non-protonized oxygen atoms of the bisulfite complex, i.e. at hydrogen bond distance from Thr199 N and at a position between the zinc ion and the hydrophobic part of the active site. The result of these and other studies have implications for our view of the catalytic function of the enzyme, since virtually all inhibitors share some features with substrate, product or expected transition states. A reaction scheme where electrophilic activation of carbon dioxide plays an important role in the hydration reaction is presented. In the reverse direction, the protonized oxygen of the bicarbonate is forced upon the zinc ion, thereby facilitating cleavage of the carbon-oxygen bond. This is achieved by the combined action of the anionic binding site, which binds carboxyl groups, the side-chain of threonine 199, which discriminates between hydrogen bond donors and acceptors, and hydrophobic interaction between substrate and the active site cavity. The required proton transfer between the zinc water and His64 can take place through water molecules 292 and 318.     

Ternary interactions of spermine with DNA: 4''-epiadriamycin and other DNA: anthracycline complexes.

The recently developed anthracycline 4'-epiadriamycin, an anti-cancer drug with improved activity, differs from adriamycin by inversion of the stereochemistry at the 4'-position. We have cocrystallized 4'-epiadriamycin with the DNA hexamer d(CGATCG) and solved the structure to 1.5 A resolution using x-ray crystallography. One drug molecule binds at each d(CG) step of the hexamer duplex. The anthracycline sugar binds in the minor groove. A feature of this complex which distinguishes it from the earlier DNA:adriamycin complex is a direct hydrogen bond from the 4'-hydroxyl group of the anthracycline sugar to the adenine N3 on the floor of the DNA minor groove. This hydrogen bond results directly from inversion of the stereochemistry at the 4'-position. Spermine molecules bind in the major groove of this complex. In anthracycline complexes with d(CGATCG) a spermine molecule binds to a continuous hydrophobic zone formed by the 5-methyl and C6 of a thymidine, C5 and C6 of a cytidine and the chromophore of the anthracycline. This report discusses three anthracycline complexes with d(CGATCG) in which the spermine molecules have different conformations yet form extensive van der Waals contacts with the same hydrophobic zone. Our results suggest that these hydrophobic interactions of spermine are DNA sequence specific and provide insight into the question of whether DNA:spermine complexes are delocalized and dynamic or site-specific and static.     

Thermodynamics of hydrogen bond and hydrophobic interactions in cyclodextrin complexes.

Values of K, delta G(o), delta H(o), delta S(o) and delta C(po) for the binding reaction of small organic ligands forming 1:1 complexes with either alpha- or beta-cyclodextrin were obtained by titration calorimetry from 15 degrees C to 45 degrees C. A hydrogen bond or hydrophobic interaction was introduced by adding a single functional group to the ligand. The thermodynamics of binding with and without the added group are compared to estimate the contribution of the hydrogen bond or hydrophobic interaction. A change in the environment of a functional group is required to influence the binding thermodynamics, but molecular size-dependent solute-solvent interactions have no effect. For phenolic O-H-O hydrogen bond formation, delta H(o) varies from -2 to -1.4 kcal mol(-1) from 15 degrees C to 45 degrees C, and delta C(p) is increased by 18 cal K(-1) mol(-1). The hydrophobic interaction has an opposite effect: in alpha-cyclodextrin, delta C(po) = -13.3 cal K(-1) mol(-1) per ligand -CH(2)-, identical to values found for the transfer of a -CH(2)-group from water to a nonpolar environment. At room temperature, the hydrogen bond and the -CH(2)-interaction each contribute about -600 cal mol(-1) to the stability (delta G(o)) of the complex. With increased temperature, the hydrogen bond stability decreases (i.e., hydrogen bonds "melt"), but the stability of the hydrophobic interaction remains essentially constant.