Favorable Entropy Of Binding Research Articles

Entropy-Enthalpy Compensation in the Methyl 5-Thio-α-D-galactopyranoside–Jacalin Interaction

Methyl 5-thio-α-D-galactopyranoside was synthesized and found to have a more favorable enthalpy of binding to Jacalin than methyl α-D-galactopyranoside, which is attributed to the greater magnitude of sulfur-π over oxygen-π interactions. This increase in enthalpy, however, was offset by a less favorable entropy of binding, arising from the need to constrain the more flexible thiosugar, thereby highlighting the complexities inherent in the design of effective sugar mimetics.

Multivalent lectin-carbohydrate interactions: Energetics and mechanisms of binding.

The biological signaling properties of lectins, which are carbohydrate-binding proteins, are due to their ability to bind and cross-link multivalent glycoprotein receptors on the surface of normal and transformed cells. While the cross-linking properties of lectins with multivalent carbohydrates and glycoproteins are relatively well understood, the mechanisms of binding of lectins to multivalent glycoconjugates are less well understood. Recently, the thermodynamics of binding of lectins to synthetic clustered glycosides, a multivalent globular glycoprotein, and to linear glycoproteins (mucins) have been described. The results are consistent with a dynamic binding mechanism in which lectins bind and jump from carbohydrate to carbohydrate epitope in these molecules. Importantly, the mechanism of binding of lectins to mucins is similar to that for a variety of protein ligands binding to DNA. Recent analysis also shows that high-affinity lectin-mucin cross-linking interactions are driven by favorable entropy of binding that is associated with the bind and jump mechanism. The results suggest that the binding of ligands to biopolymers, in general, may involve a common mechanism that involves enhanced entropic effects which facilitate binding and subsequent complex formation including enzymology.

Biophysical Characterization of the Contribution of the Fab Region to the IgG-FcγRIIIa Interaction.

The cell-surface receptor FcγRIIIa is crucial to the efficacy of therapeutic antibodies as well as the immune response. The interaction of the Fc region of IgG molecules with FcγRIIIa has been characterized, but until recently, it was thought that the Fab regions were not involved in the interaction. To evaluate the influence of the Fab regions in a biophysical context, we carried out surface plasmon resonance analyses using recombinant FcγRIIIa ligands. A van't Hoff analysis revealed that compared to the interaction of the papain-digested Fc fragment with FcγRIIIa, the interaction of commercially available, full-length rituximab with FcγRIIIa had a more favorable binding enthalpy, a less favorable binding entropy, and a slower off rate. Similar results were obtained from analyses of IgG1 molecules and an IgG1-Fc fragment produced by Expi293 cells. For further validation, we also prepared a maltose-binding protein-linked IgG1-Fc fragment (MBP-Fc). The binding enthalpy of MBP-Fc was nearly equal to that of the IgG1-Fc fragment for the interaction with FcγRIIIa, indicating that such alternatives to the Fab domains as MBP do not positively contribute to the IgG-FcγRIIIa interactions. Our investigation strongly suggests that the Fab region directly interacts with FcγRIIIa, resulting in an increase in the binding enthalpy and a decrease in the dissociation rate, at the expense of favorable binding entropy.

Mechanism of Mucin Recognition by Lectins: A Thermodynamic Study.

Isothermal titration microcalorimetry (ITC) can directly determine the thermodynamic binding parameters of biological molecules including affinity constant, binding stoichiometry, heat of binding (enthalpy) and indirectly the entropy, and free energy of binding. ITC has been extensively used to study the binding of lectins to mono- and oligosaccharides, but limitedly in applications to lectin-glycoprotein interactions. Inherent experimental challenges to ITC include sample precipitation during the experiment and relative high amount of sample required, but careful design of experiments can minimize these problems and allow valuable information to be obtained. For example, the thermodynamics of binding of lectins to multivalent globular and linear glycoproteins (mucins) have been described. The results are consistent with a dynamic binding mechanism in which lectins bind and jump from carbohydrate to carbohydrate epitope in these molecules leading to increased affinity. Importantly, the mechanism of binding of lectins to mucins appears similar to that for a variety of protein ligands binding to DNA. Recent results also show that high-affinity lectin-mucin cross-linking interactions are driven by favorable entropy of binding that is associated with the bind and jump mechanism. The results suggest that the binding of ligands to biopolymers, in general, may involve a common mechanism that involves enhanced entropic effects that facilitate binding interactions.

Repositioning of the Anthelmintic Drugs Bithionol and Triclabendazole as Transthyretin Amyloidogenesis Inhibitors.

Transthyretin (TTR) is a causative protein of TTR amyloidosis (ATTR amyloidosis), a general term for diseases characterized by deposition of TTR amyloid fibrils in specific organs. ATTR amyloidosis can be ameliorated by stabilization of the TTR tetramer through the binding of small molecules. Here, we show that the clinical anthelmintic drugs bithionol (42) and triclabendazole (43) potently inhibit aggregation of the amyloidogenic variant V30M-TTR. A competitive binding assay using a fluorescence probe showed that the binding affinity of 42 with V30M-TTR was significantly higher than that of the first-in-class drug tafamidis (1), and the binding affinity of 43 was similar to that of 1. The crystallographic and thermodynamic analysis revealed that 42 efficiently occupied the halogen-binding grooves of TTR, resulting in the favorable binding entropy. Multifaceted in vitro studies of anthelmintic drugs have the potential to reposition these drugs as ATTR amyloidosis inhibitors.

Structure-thermodynamics-relationships of hepatitis C viral NS3 protease inhibitors

Thermodynamic parameters were determined for structurally-related inhibitors of HCV NS3 protease to assess how binding entropies and enthalpies vary with incremental changes at the P2 and P3 inhibitor subsites. Changing the heterocyclic substituent at P2 from a pyridyl to a 7-methoxy-2-phenyl-4-quinolyl group leads to a 710-fold increase in affinity. Annelating a benzene ring onto a pyridine ring leads to quinoline-derived inhibitors having higher affinities, but the individual enthalpy and entropy contributions are markedly different for each ligand pair. Introducing a phenyl group at C2 of the heterocyclic ring at P2 uniformly leads to higher affinity analogs with more favorable binding entropies, while adding a methoxy group at C7 of the quinoline ring at P2 provides derivatives with more favorable binding enthalpies. Significant enthalpy/entropy compensation is observed for structural changes made to inhibitors lacking a 2-phenyl substituent, whereas favorable changes in both binding enthalpies and entropies accompany structural modifications when a 2-phenyl group is present. Overall, binding energetics of inhibitors having a 2-phenyl-4-quinolyl group at P2 are dominated by entropic effects, whereas binding of the corresponding norphenyl analogs are primarily enthalpy driven. Notably, the reversal from an entropy driven association to an enthalpy driven one for this set of inhibitors also correlates with alternate binding modes. When the steric bulk of the side chain at P3 is increased from a hydrogen atom to a tert-butyl group, there is a 770-fold improvement in affinity. The 30-fold increase resulting from the first methyl group is solely the consequence of a more favorable change in entropy, whereas subsequent additions of methyl groups leads to modest increases in affinity that arise primarily from incremental improvements in binding enthalpies accompanied with smaller favorable entropic contributions.

Shift from Entropic Cu2+ Binding to Enthalpic Cu+ Binding Determines the Reduction Thermodynamics of Blue Copper Proteins.

The enthalpic and entropic components of Cu2+ and Cu+ binding to the blue copper protein azurin have been quantified with isothermal titration calorimetry (ITC) measurements and analysis, providing the first such experimental values for Cu+ binding to a protein. The high affinity of azurin for Cu2+ is entirely due to a very favorable binding entropy, while its even higher affinity for Cu+ is due to a favorable binding enthalpy and entropy. The binding thermodynamics provide insight into bond enthalpies at the blue copper site and entropic contributions from desolvation and proton displacement. These values were used in thermodynamic cycles to determine the enthalpic and entropic contributions to the free energy of reduction and thus the reduction potential. The reduction thermodynamics obtained with this method are in good agreement with previous results from temperature-dependent electrochemical measurements. The calorimetry method, however, provides new insight into contributions from the initial (oxidized) and final (reduced) states of the reduction. Since ITC measurements quantify the protons that are displaced upon metal binding, the proton transfer that is coupled with electron transfer is also determined with this method. Preliminary results for Cu2+ and Cu+ binding to the Phe114Pro variant of azurin demonstrate the insight about protein tuning of the reduction potential that is provided by the binding thermodynamics of each metal oxidation state.

Protein-ligand binding enthalpies from near-millisecond simulations: Analysis of a preorganization paradox.

Calorimetric studies of protein-ligand binding sometimes yield thermodynamic data that are difficult to understand. Today, molecular simulations can be used to seek insight into such calorimetric puzzles, and, when simulations and experiments diverge, the results can usefully motivate further improvements in computational methods. Here, we apply near-millisecond duration simulations to estimate the relative binding enthalpies of four peptidic ligands with the Grb2 SH2 domain. The ligands fall into matched pairs, where one member of each pair has an added bond that preorganizes the ligand for binding and thus may be expected to favor binding entropically, due to a smaller loss in configurational entropy. Calorimetric studies have shown that the constrained ligands do in fact bind the SH2 domain more tightly than the flexible ones, but, paradoxically, the improvement in affinity for the constrained ligands is enthalpic, rather than entropic. The present enthalpy calculations yield the opposite trend, as they suggest that the flexible ligands bind more exothermically. Additionally, the small relative binding enthalpies are found to be balances of large differences in the energies of structural components such as ligand and the binding site residues. As a consequence, the deviations from experiment in the relative binding enthalpies represent small differences between these large numbers and hence may be particularly susceptible to error, due, for example, to approximations in the force field. We also computed first-order estimates of changes in configurational entropy on binding. These too are, arguably, paradoxical, as they tend to favor binding of the flexible ligands. The paradox is explained in part by the fact that the more rigid constrained ligands reduce the entropy of binding site residues more than their flexible analogs do, at least in the simulations. This result offers a rather general counterargument to the expectation that preorganized ligands should be associated with more favorable binding entropies, other things being equal.

Entropy as a Driver of Selectivity for Inhibitor Binding to Histone Deacetylase 6.

Among the metal-dependent histone deacetylases, the class IIb isozyme HDAC6 is remarkable because of its role in the regulation of microtubule dynamics in the cytosol. Selective inhibition of HDAC6 results in microtubule hyperacetylation, leading to cell cycle arrest and apoptosis, which is a validated strategy for cancer chemotherapy and the treatment of other disorders. HDAC6 inhibitors generally consist of a Zn2+-binding group such as a hydroxamate, a linker, and a capping group; the capping group is a critical determinant of isozyme selectivity. Surprisingly, however, even "capless" inhibitors exhibit appreciable HDAC6 selectivity. To probe the chemical basis for this selectivity, we now report high-resolution crystal structures of HDAC6 complexed with capless cycloalkyl hydroxamate inhibitors 1-4. Each inhibitor hydroxamate group coordinates to the catalytic Zn2+ ion with canonical bidentate geometry. Additionally, the olefin moieties of compounds 2 and 4 bind in an aromatic crevice between the side chains of F583 and F643. Reasoning that similar binding could be achieved in the representative class I isozyme HDAC8, we employed isothermal titration calorimetry to study the thermodynamics of inhibitor binding. These measurements indicate that the entropy of inhibitor binding is generally positive for binding to HDAC6 and negative for binding to HDAC8, resulting in ≤313-fold selectivity for binding to HDAC6 relative to HDAC8. Thus, favorable binding entropy contributes to HDAC6 selectivity. Notably, cyclohexenyl hydroxamate 2 represents a promising lead for derivatization with capping groups that may further enhance its impressive 313-fold thermodynamic selectivity for HDAC6 inhibition.

Enthalpy-Entropy Compensation in the Binding of Modulators at Ionotropic Glutamate Receptor GluA2

The 1,2,4-benzothiadiazine 1,1-dioxide type of positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) are promising lead compounds for the treatment of cognitive disorders, e.g., Alzheimer’s disease. The modulators bind in a cleft formed by the interface of two neighboring ligand binding domains and act by stabilizing the agonist-bound open-channel conformation. The driving forces behind the binding of these modulators can be significantly altered with only minor substitutions to the parent molecules. In this study, we show that changing the 7-fluorine substituent of modulators BPAM97 (2) and BPAM344 (3) into a hydroxyl group (BPAM557 (4) and BPAM521 (5), respectively), leads to a more favorable binding enthalpy (ΔH, kcal/mol) from −4.9 (2) and −7.5 (3) to −6.2 (4) and −14.5 (5), but also a less favorable binding entropy (−TΔS, kcal/mol) from −2.3 (2) and −1.3 (3) to −0.5 (4) and 4.8 (5). Thus, the dissociation constants (Kd, μM) of 4 (11.2) and 5 (0.16) are similar to those of 2 (5.6) and 3 (0.35). Functionally, 4 and 5 potentiated responses of 10 μM L-glutamate at homomeric rat GluA2(Q)i receptors with EC50 values of 67.3 and 2.45 μM, respectively. The binding mode of 5 was examined with x-ray crystallography, showing that the only change compared to that of earlier compounds was the orientation of Ser-497 pointing toward the hydroxyl group of 5. The favorable enthalpy can be explained by the formation of a hydrogen bond from the side-chain hydroxyl group of Ser-497 to the hydroxyl group of 5, whereas the unfavorable entropy might be due to desolvation effects combined with a conformational restriction of Ser-497 and 5. In summary, this study shows a remarkable example of enthalpy-entropy compensation in drug development accompanied with a likely explanation of the underlying structural mechanism.

Molecular Mechanisms of Alzheimer's Biomarker FDDNP Binding to Aβ Amyloid Fibril.

Using isobaric-isothermal replica exchange molecular dynamics and the all-atom explicit water model, we examined the binding of FDDNP biomarkers to the Aβ amyloid fibril fragment. Our results can be summarized as follows. First, FDDNP ligands bind with high affinity to the Aβ fibril, and the hydrophobic effect together with π-stacking interactions are the dominant factors governing FDDNP binding. In comparison, electrostatic interactions and hydrogen bonding play a minor role. Second, our simulations reveal a strong tendency of bound FDDNP molecules for self-aggregation. Accordingly, about two-thirds of all bound ligands form aggregated clusters of various sizes, and ligand-ligand interactions make considerable contribution to FDDNP binding. Third, FDDNP ligands bind to two distinct sites on the Aβ fibril. Primary binding sites (NT) are located at the N-terminals of Aβ10-40 peptides, whereas secondary ones (CE) occur on the concave fibril edge near fibril channels. The NT sites are characterized by strong hydrophobic and π-stacking interactions, favorable binding entropy resulting from multiple FDDNP binding orientations and propensity for self-aggregation but relatively weak van der Waals interactions. In contrast, the CE sites offer stronger van der Waals binding interactions but weaker hydrophobic and aromatic interactions and less favorable binding entropy. By comparing our data with previous studies, we suggest that the primary binding locations identified by us are likely to occur in other Aβ fibril polymorphic structures. We also show that FDDNP binds via distinct mechanisms to Aβ fibrils and monomers. We argue that FDDNP binds with stronger affinity to benign Aβ monomers than to the fibrils, raising questions about the ability of FDDNP to selectively label amyloid deposits.

A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility.

Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.

Probing lectin-mucin interactions by isothermal titration microcalorimetry.

Isothermal titration microcalorimetry (ITC) can directly determine the thermodynamic binding parameters of biological molecules including affinity constant, binding stoichiometry, and heat of binding (enthalpy) and indirectly the entropy and free energy of binding. ITC has been extensively used to study the binding of lectins to mono- and oligosaccharides, but limited applications to lectin-glycoprotein interactions. Inherent experimental challenges to ITC include sample precipitation during the experiment and relative high amount of sample required, but careful design of experiments can minimize these problems and allow valuable information to be obtained. For example, the thermodynamics of binding of lectins to multivalent globular and linear glycoproteins (mucins) have been described. The results are consistent with a dynamic binding mechanism in which lectins bind and jump from carbohydrate to carbohydrate epitope in these molecules leading to increased affinity. Importantly, the mechanism of binding of lectins to mucins appears similar to that for a variety of protein ligands binding to DNA. Recent results also show that high affinity lectin-mucin cross-linking interactions are driven by favorable entropy of binding that is associated with the bind and jump mechanism. The results suggest that the binding of ligands to biopolymers, in general, may involve a common mechanism that involves enhanced entropic effects that facilitate binding interactions.

Probing the effect of conformational constraint on phosphorylated ligand binding to an SH2 domain using polarizable force field simulations.

Preorganizing a ligand in the conformation it adopts upon binding to a protein has long been considered to be an effective way to improve affinity by making the binding entropy more favorable. However, recent thermodynamic studies of a series of complexes of the Grb2 SH2 domain with peptide analogues having constrained and flexible replacements for a phosphotyrosine residue revealed that less favorable binding entropies may result from constraining ligands in their biologically active conformations. Toward probing the origin of this unexpected finding, we examined the complexes of four phosphotyrosine-derived analogues with the Grb2 SH2 domain using molecular dynamics simulations with a polarizable force field. Significantly, the computed values for the relative binding free energies, entropies, and enthalpies of two pairs of constrained and unconstrained ligands reproduced the trends that were determined experimentally, although the relative differences were overestimated. These calculations also revealed that a large fraction of the ligands lacking the constraining element exist in solution as compact, macrocyclic-like structures that are stabilized by interactions between the phosphate groups and the amide moieties of the C-terminal pY+2 residues. In contrast, the three-membered ring in the constrained ligands prevents the formation of such macrocyclic structures, leading instead to globally extended, less ordered conformations. Quasiharmonic analysis of these conformational ensembles suggests that the unconstrained ligands possess significantly lower entropies in solution, a finding that is consistent with the experimental observation that the binding entropies for the unconstrained ligands are more favorable than for their constrained counterparts. This study suggests that introducing local constraints in flexible molecules may have unexpected consequences, and a detailed understanding of the conformational preferences of ligands in their unbound states is a critical prerequisite to correlating changes in their chemical structure with protein binding entropies and enthalpies.

Determining the Role of the Aromatic Ring of N‐Arylmethyl ent‐conduramine F‐1 in their Interactions with α‐Glucosidases by Saturation Transfer Difference NMR Spectroscopy Experiments

Glucosidases are a group of enzymes responsible of the glycosidic bond cleavage with different specificities depending on the number of monosaccharides, the position of cleavage, and the configuration of the hydroxy groups in their substrates. In particular, inhibitors of α-1,4-glucosidases have generated great interest as potential therapeutic agents for the treatment of type II diabetes (e.g., miglitol (N-2-hydroxyethyl-1-deoxynojirimycin), Glyset, Diastabol, Glucobay),1, 2 obesity,3 hepatitis B and C4, 5 and other viral diseases,6 and cancer.7

Protein–Ligand Interactions: Thermodynamic Effects Associated with Increasing Nonpolar Surface Area

Thermodynamic parameters were determined for complex formation between the Grb2 SH2 domain and Ac-pTyr-Xaa-Asn derived tripeptides in which the Xaa residue is an α,α-cycloaliphatic amino acid that varies in ring size from three- to seven-membered. Although the six- and seven-membered ring analogs are approximately equipotent, binding affinities of those having three- to six-membered rings increase incrementally with ring size because increasingly more favorable binding enthalpies dominate increasingly less favorable binding entropies, a finding consistent with an enthalpy-driven hydrophobic effect. Crystallographic analysis reveals that the only significant differences in structures of the complexes are in the number of van der Waals contacts between the domain and the methylene groups in the Xaa residues. There is a positive correlation between buried nonpolar surface area and binding free energy and enthalpy, but not with ΔC(p). Displacing a water molecule from a protein-ligand interface is not necessarily reflected in a favorable change in binding entropy. These findings highlight some of the fallibilities associated with commonly held views of relationships of structure and energetics in protein-ligand interactions and have significant implications for ligand design.

NMR and amber analysis of the neamine pharmacophore for the design of novel aminoglycoside antibiotics

The dependence of the solution structure of neamine on pH was determined by NMR and AMBER molecular dynamics methods at pD 3.3, pD 6.5, and pD 7.4 in D 2O at 25 °C. Unlike neamine structures at pD 3.3 and 6.5, which essentially showed only one conformer, slowly exchanging primary, P-state, and secondary, S-state, neamine conformers populated on the NMR time scale at ∼80% and ∼20%, respectively, were detected at pD 7.4 with kinetic constants k on(P→S) = 1.9771 s −1 and k off(S→P) = 1.1319 s −1. A tertiary, T-state, neamine species populated at ∼3% was also detected by NMR at pD 7.4. The p Ka values determined by NMR titration experiments are p Ka1 6.44 ± 0.13 for N3 of ring-II, p Ka2 7.23 ± 0.09 for N2′ of ring-I, p Ka3 7.77 ± 0.19 for N1 of ring-II, and p Ka4 8.08 ± 0.15 for N6′ of ring-I. Ring-I and ring-II of the P-state neamine and ring-I of the S and T-states of neamine possess the 4C 1 chair conformation between pD 3.3 and pD = 7.4. In contrast, ring-II of the S and T-states of neamine most likely adopt the 6rH 1 half-chair conformation. The P and S-states of neamine exhibit a negative syn-ψ glycosidic geometry. The exocyclic aminomethyl group of ring-I adopts the gt exocyclic rotamer conformation around physiological pHs while the gg exocyclic rotamer conformation predominates in acidic solutions near and below pH 4.5. Neamine exists in the P-state as a mixture of tetra-/tri-/di-protonated species between pD 4.5 and pD 7.4, while the S-state neamine exist only in a di-protonated species around physiological pDs. The existence of the S-state neamine may facilitate binding of neamine-like aminoglycosides by favorable entropy of binding to the A-site of 16S ribosomal RNA, suggesting that novel aminoglycoside compounds carrying a S-state neamine pharmacophore can be developed.

Thermodynamic and Structural Effects of Macrocyclic Constraints in Protein−Ligand Interactions

The thermodynamic and structural effects of macrocyclization as a tactic for stabilizing the biologically-active conformation of Grb2 SH2 binding peptides were investigated using isothermal titration calorimetry and x-ray crystallography. 23-Membered macrocycles containing the sequence pYVN were slightly more potent than their linear controls; however, preorganization did not necessarily eventuate in a more favorable binding entropy. Structures of complexes of macrocycle 7 and its acyclic control 8 are similar except for differences in relative orientations of corresponding atoms in the linking moieties of 7 and 8. There are no differences in the number of direct or water-mediated protein-ligand contacts that might account for the less favorable binding enthalpy of 7; however, an intramolecular hydrogen bond between the pY and pY+3 residues in 8 that is absent in 7 may be a factor. These studies highlight the difficulties associated with correlating energetics and structure in protein-ligand interactions.

Altered Enthalpy−Entropy Compensation in Picomolar Transition State Analogues of Human Purine Nucleoside Phosphorylase

Human purine nucleoside phosphorylase (PNP) belongs to the trimeric class of PNPs and is essential for catabolism of deoxyguanosine. Genetic deficiency of PNP in humans causes a specific T-cell immune deficiency, and transition state analogue inhibitors of PNP are in development for treatment of T-cell cancers and autoimmune disorders. Four generations of Immucillins have been developed, each of which contains inhibitors binding with picomolar affinity to human PNP. Full inhibition of PNP occurs upon binding to the first of three subunits, and binding to subsequent sites occurs with negative cooperativity. In contrast, substrate analogue and product bind without cooperativity. Titrations of human PNP using isothermal calorimetry indicate that binding of a structurally rigid first-generation Immucillin (K(d) = 56 pM) is driven by large negative enthalpy values (DeltaH = -21.2 kcal/mol) with a substantial entropic (-TDeltaS) penalty. The tightest-binding inhibitors (K(d) = 5-9 pM) have increased conformational flexibility. Despite their conformational freedom in solution, flexible inhibitors bind with high affinity because of reduced entropic penalties. Entropic penalties are proposed to arise from conformational freezing of the PNP.inhibitor complex with the entropy term dominated by protein dynamics. The conformationally flexible Immucillins reduce the system entropic penalty. Disrupting the ribosyl 5'-hydroxyl interaction of transition state analogues with PNP causes favorable entropy of binding. Tight binding of the 17 Immucillins is characterized by large enthalpic contributions, emphasizing their similarity to the transition state. Via introduction of flexibility into the inhibitor structure, the enthalpy-entropy compensation pattern is altered to permit tighter binding.

Free Energies and Entropies of Water Molecules at the Inhibitor−Protein Interface of DNA Gyrase

Complexes of the antibiotics novobiocin and clorobiocin with DNA gyrase are illustrative of the importance of bound water to binding thermodynamics. Mutants resistant to novobiocin as well as those with a decreased affinity for novobiocin over clorobiocin both involve a less favorable entropy of binding, which more than compensates for a more favorable enthalpy, and additional water molecules at the protein-ligand interface. Free energy, enthalpy, and entropy for these water molecules were calculated by thermodynamic integration computer simulations. The calculations show that addition of the water molecules is entropically unfavorable, with values that are comparable to the measured entropy differences. The free energies and entropies correlate with the change in the number of hydrogen bonds due to the addition of water molecules.