Structure-activity relationships in engineered proteins: characterization of disruptive deletions in the alpha-ammonium group binding site of tyrosyl-tRNA synthetase

Residues Asp-78 and Gln-173 of the tyrosyl-tRNA synthetase of Bacillus stearothermophilus form part of the binding site for tyrosine by making hydrogen bonds with the alpha-ammonium group. Asp-38 is close enough to the group to make an important electrostatic contribution. Unlike other residues in the active site that have been studied by site-directed mutagenesis, Asp-38, Asp-78, and Gln-173 are part of hydrogen-bonded networks. Each of these residues has been mutated to an alanine, and the resultant mutants have been studied by kinetics to construct the difference energy diagrams for the formation of tyrosyl adenylate. In each example, the binding of tyrosine is weakened by about 2.5 kcal mol-1. But, unlike previous mutants, the dissociation of the second substrate, in this case ATP, is also seriously affected, being weakened by some 2 kcal mol-1 for TyrTS(Ala-78) and TyrTS(Ala-173). The energy of the transition state for the formation of tyrosyl adenylate is raised by 7.8 kcal mol-1 for the former and 4.5 kcal mol-1 for the latter mutant. Addition of these mutants to linear free energy plots constructed for the nondisruptive mutants in the accompanying study [Fersht, A. R., Leatherbarrow, R. J., & Wells, T. N. C. (1987) Biochemistry (preceding paper in this issue)] reveals large deviations of the data for TyrTS(Ala-38) and TyrTS(Ala-78) from the regression line. These thus belong to a different class of mutations from previous nondisruptive examples. This observation combined with the structural evidence and difference energy diagrams strongly suggests that the mutations Asp----Ala-38 and Asp----Ala-78 are disruptive in nature.     

Catalysis and linear free energy relationships in aspartic proteases

Aspartic proteases are receiving considerable attention as potential drug targets in several serious diseases, such as AIDS, malaria, and Alzheimer's disease. These enzymes cleave polypeptide chains, often between specific amino acid residues, but despite the common reaction mechanism, they exhibit large structural differences. Here, the catalytic mechanism of aspartic proteases plasmepsin II, cathepsin D, and HIV-1 protease is examined by computer simulations utilizing the empirical valence bond approach in combination with molecular dynamics and free energy perturbation calculations. Free energy profiles are established for four different substrates, each six amino acids long and containing hydrophobic side chains in the P1 and P1' positions. Our simulations reproduce the catalytic effect of these enzymes, which accelerate the reaction rate by a factor of approximately 10(10) compared to that of the corresponding uncatalyzed reaction in water. The calculations elucidate the origin of the catalytic effect and allow a rationalization of the fact that, despite large structural differences between plasmepsin II/cathepsin D and HIV-1 protease, the magnitude of their rate enhancement is very similar. Amino acid residues surrounding the active site together with structurally conserved water molecules are found to play an important role in catalysis, mainly through dipolar (electrostatic) stabilization. A linear free energy relationship for the reactions in the different enzymes is established that also demonstrates the reduced reorganization energy in the enzymes compared to that in the uncatalyzed water reaction.     

Linear free energy relationships in enzyme binding interactions studied by protein engineering.

Experiments on mutants of tyrosyl-tRNA synthetase have shown that there can be linear free energy relationships (LFERs) between changes in activation free energies and changes in binding energies when groups are deleted that bind to non-reacting parts of the substrate (Fersht et al., 1986, 1987). It has now been proposed (Straub and Karplus, 1990) that such LFERs can occur for the mutation of hydrogen bonding groups only for the limiting examples of Br?nsted beta of 0, 1 or infinity, and that fractional values of beta are not permissible. The reasoning behind this is that the energy of a hydrogen bond is not linear with distance and the (false) premise that an LFER requires that there is a linear relationship between bond energy and distance. We show from a simple model how LFERs can arise for binding interactions and how they can give fractional values of beta, in accord with experimental evidence. An LFER occurs between binding and catalysis when a set of interactions exists in which each member contributes to the binding energy of the transition state the same fraction of the binding energy it contributes to the products (both relative to the ground state).     

Total free energy analysis of fully hydrated proteins

The total free energy of a hydrated biomolecule and its corresponding decomposition of energy and entropy provides detailed information about regions of thermodynamic stability or instability. The free energies of four hydrated globular proteins with different net charges are calculated from a molecular dynamics simulation, with the energy coming from the system Hamiltonian and entropy using multiscale cell correlation. Water is found to be most stable around anionic residues, intermediate around cationic and polar residues, and least stable near hydrophobic residues, especially when more buried, with stability displaying moderate entropy-enthalpy compensation. Conversely, anionic residues in the proteins are energetically destabilized relative to singly solvated amino acids, while trends for other residues are less clear-cut. Almost all residues lose intraresidue entropy when in the protein, enthalpy changes are negative on average but may be positive or negative, and the resulting overall stability is moderate for some proteins and negligible for others. The free energy of water around single amino acids is found to closely match existing hydrophobicity scales. Regarding the effect of secondary structure, water is slightly more stable around loops, of intermediate stability around $\beta$ strands and turns, and least stable around helices. An interesting asymmetry observed is that cationic residues stabilize a residue when bonded to its N-terminal side but destabilize it when on the C-terminal side, with a weaker reversed trend for anionic residues.     

Structure-activity relationships of steroid hormones on muscarinic receptor binding

A total of fifty steroidal compounds were tested for their inhibition on the binding of muscarinic receptor antagonist, [3H]quinuclidinyl benzilate ([3H](-)QNB), to the hypothalamic membranes prepared from male rats. Among the compounds tested, the active structures (with IC50 values less than or equal to 100 microM in parentheses) are: progesterone (40), 5 beta-pregnane-3,20-dione (40), deoxy-corticosterone (50), 5 beta-pregnane-17 alpha,21-diol-3,20-dione (30), 11-desoxy-17-hydroxycorticosterone (22), 17 alpha-hydroxyprogesterone (20), 5 beta-pregnan-17 alpha-ol-3,20-dione (24), 5 beta-androstane-3,17-dione (100), and 5 beta-dihydrotestosterone (100). By examining all the compounds tested, the following structure-activity relationship became apparent: (a) The ring A-reduced steroidal structures with a 5 beta-conformation were more potent than those with a 5 alpha-conformation; (b) 17 alpha-hydroxylation of the steroidal ring increased the steroid's inhibitory activity; (c) The C3 carbonyl group was essential for activity; (d) Reduction of the C3 carbonyl group or aromatization of the ring A abolished the steroid's inhibitory activity; (e) Oxidation of the C11 position of ring C resulted in a decrease or loss of inhibitory activity; and (f) Different modifications of the side chain of ring D by acetylation resulted in either an increase or a decrease in the inhibitory activity. The structure-activity relationship as revealed in this study might provide an insight for the synthesis of a steroidal molecule with a high affinity for the muscarinic receptor as well as for the search of a more potent and physiologically relevant steroidal metabolite possessing the ability to interact with the muscarinic receptor.     

Structure-activity relationships and binding model of novel aromatase inhibitors

The use of aromatase inhibitors is an established therapy for oestrogen-dependent breast cancer in postmenopausal women. However, the sole commercially available aromatase inhibitor, aminoglutethimide, is not very selective. We have therefore developed fadrozole hydrochloride and CGS 20 267, which are both currently under clinical evaluation. This report will present an analysis of structure-activity relationships in the azole series of inhibitors and give an account of the further optimization of our development compounds, starting from CGS 20 267 over CGP 45 688 and leading to CGP 47 645, the most potent aromatase inhibitors in vivo reported to date. In addition, on the basis of comparisons of these azole-type inhibitors with the most potent steroidal inhibitors published in the literature, we propose a CAMM-generated model describing the relative binding modes of these two classes of compounds at the active site of the enzyme.     

Protection of an unstable reaction intermediate examined with linear free energy relationships in tyrosyl-tRNA synthetase

Linear free energy relationships (LFERs) are powerful tools in the search to understand the relationship between molecular structure and activity. They frequently link the changes in the rate constants for a reaction to changes in the equilibrium constant caused by alterations in structure. In physical-organic chemistry, these have been interpreted to give information on the structure of the transition state. Similar phenomena have been observed for reactions catalyzed by a series of engineered mutants of tyrosyl-tRNA synthetase from Bacillus stearothermophilus. LFERs are applied in this study to probe how the enzyme minimizes its side reactions. A linear free energy relationship is shown between the binding of the unstable enzyme-tyrosyl adenylate complex and its rate constant of hydrolysis. However, mutations of a key residue, His48, show significant deviation from the relationship, implying a role for the side chain in protection of the complex from hydroxide attack. A second linear free energy relationship is shown linking the rate and equilibrium constants for tyrosyl adenylate binding to the enzyme. Four distinct classes of mutation are discussed in the context of this relationship. The data from all but one of these groups of mutations conform well to a linear free energy relationship between the dissociation rate and dissociation equilibrium constants for the enzyme-tyrosyl adenylate complex with slope beta = 1.01 +/- 0.08. The specificity of binding of tyrosyl adenylate is determined solely by its dissociation rate constant of the intermediate, and the mutations have relatively little effect on the enzyme-tyrosyl adenylate association rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombin inhibitors with novel P1 binding pocket functionality: free energy of binding analysis

The high incidence of thrombembolic diseases justifies the development of new antithrombotics. The search for a direct inhibitor has resulted in the synthesis of a considerable number of low molecular weight molecules that inhibit human α-thrombin potently. However, efforts to develop an orally active drug remain in progress as the most active inhibitors with a highly basic P1 moiety exhibit an unsatisfactory bioavailability profile. In our previous work we solved several X-ray structures of human α-thrombin in complexes with (1) novel bicyclic arginine mimetics attached to the glycylproline amide and pyridinone acetamide scaffold and (2) inhibitors with a novel aza scaffold and with charged or neutral P1 moieties. In the present contribution, we correlate the structures of the complex between these inhibitors and the protein with the calculated free energy of binding. The energy of solvation was calculated using the Poisson–Boltzmann approach. In particular, the requirements for successful recognition of an inhibitor at the protein’s active site pocket S1 are discussed. Figure We report here on free energy of binding analysis of thrombin inhibitors with novel aza scaffold and novel bicyclic arginine mimetics in S1 pocket of thrombin     

Protein free energy landscapes remodeled by ligand binding

Glucose/galactose binding protein (GGBP) functions in two different larger systems of proteins used by enteric bacteria for molecular recognition and signaling. Here we report on the thermodynamics of conformational equilibrium distributions of GGBP. Three fluorescence components appear at zero glucose concentration and systematically transition to three components at high glucose concentration. Fluorescence anisotropy correlations, fluorescent lifetimes, thermodynamics, computational structure minimization, and literature work were used to assign the three components as open, closed, and twisted conformations of the protein. The existence of three states at all glucose concentrations indicates that the protein continuously fluctuates about its conformational state space via thermally driven state transitions; glucose biases the populations by reorganizing the free energy profile. These results and their implications are discussed in terms of the two types of specific and nonspecific interactions GGBP has with cytoplasmic membrane proteins.     

Insights into protein-protein binding by binding free energy calculation and free energy decomposition for the Ras-Raf and Ras-RalGDS complexes

Absolute binding free energy calculations and free energy decompositions are presented for the protein-protein complexes H-Ras/C-Raf1 and H-Ras/RalGDS. Ras is a central switch in the regulation of cell proliferation and differentiation. In our study, we investigate the capability of the molecular mechanics (MM)-generalized Born surface area (GBSA) approach to estimate absolute binding free energies for the protein-protein complexes. Averaging gas-phase energies, solvation free energies, and entropic contributions over snapshots extracted from trajectories of the unbound proteins and the complexes, calculated binding free energies (Ras-Raf: -15.0(+/-6.3)kcal mol(-1); Ras-RalGDS: -19.5(+/-5.9)kcal mol(-1)) are in fair agreement with experimentally determined values (-9.6 kcal mol(-1); -8.4 kcal mol(-1)), if appropriate ionic strength is taken into account. Structural determinants of the binding affinity of Ras-Raf and Ras-RalGDS are identified by means of free energy decomposition. For the first time, computationally inexpensive generalized Born (GB) calculations are applied in this context to partition solvation free energies along with gas-phase energies between residues of both binding partners. For selected residues, in addition, entropic contributions are estimated by classical statistical mechanics. Comparison of the decomposition results with experimentally determined binding free energy differences for alanine mutants of interface residues yielded correlations with r(2)=0.55 and 0.46 for Ras-Raf and Ras-RalGDS, respectively. Extension of the decomposition reveals residues as far apart as 25A from the binding epitope that can contribute significantly to binding free energy. These "hotspots" are found to show large atomic fluctuations in the unbound proteins, indicating that they reside in structurally less stable regions. Furthermore, hotspot residues experience a significantly larger-than-average decrease in local fluctuations upon complex formation. Finally, by calculating a pair-wise decomposition of interactions, interaction pathways originating in the binding epitope of Raf are found that protrude through the protein structure towards the loop L1. This explains the finding of a conformational change in this region upon complex formation with Ras, and it may trigger a larger structural change in Raf, which is considered to be necessary for activation of the effector by Ras.     

Ferric uptake regulator protein: binding free energy calculations and per-residue free energy decomposition

Iron homeostasis is, in many bacterial species, mediated by the ferric uptake regulator (Fur). A regulatory site able to bind iron to activate Fur for DNA binding has been described, and a structural zinc site essential for the dimerization has also been proposed. They have been localized and named site 1 and site 2, respectively, from the crystal structure of a zinc-substituted Pseudomonas aeruginosa Fur (PA-Fur). Notwithstanding the studies on Fur proteins from various species, both the precise site of iron binding and the effect on DNA binding affinity are still controversial. These issues were investigated here by molecular dynamics simulations and free energy calculations. Simulations were performed for eight molecular systems represented by the three forms of Fur, that is, apo Fur, metal-substituted Fur, and Fur complexed with DNA. Because of the lack of a Fur-DNA complex crystal structure, the recently published model based on mass spectrometry experiments on Escherichia coli Fur (EC-Fur), and the crystal structure of PA-Fur, was used, after adjustment to adopt a symmetric conformation. The simulation results suggest that the formerly proposed site 2 is, in fact, the regulatory iron-sensing site. The calculations also predict that Fe(2+) at site 2 is hexacoordinated having an octahedral environment with only nitrogen and oxygen atoms, which is in accordance with previous spectroscopic characterizations. Energy decomposition pinpoints H87 as an additional amino acid that defines the regulatory metal site. Finally, free energy decomposition analysis reveals a number of amino acids potentially important in dimerization and in DNA binding.     

Structure-activity relationships of linear and cyclic peptides containing the NGR tumor-homing motif

Cyclic and linear peptides containing the Asn-Gly-Arg (NGR) motif have proven useful for delivering various anti-tumor compounds and viral particles to tumor vessels. We have investigated the role of cyclic constraints on the structure and tumor-homing properties of NGR peptides using tumor necrosis factor-alpha (TNF) derivatives containing disulfide-bridged (CNGRC-TNF) and linear (GNGRG-TNF) NGR domains. Experiments carried out in animal models showed that both GNGRG and CNGRC can target TNF to tumors. However, the anti-tumor activity of CNGRC-TNF was >10-fold higher than that of GNGRG-TNF. Molecular dynamic simulation of cyclic CNGRC showed the presence of a bend geometry involving residues Gly(3)-Arg(4). Molecular dynamic simulation of the same peptide without disulfide constraints showed that the most populated and thermodynamically favored configuration is characterized by the presence of a beta-turn involving residues Gly(3)-Arg(4) and hydrogen bonding interactions between the backbone atoms of Asn(2) and Cys(5). These results suggest that the NGR motif has a strong propensity to form beta-turn in linear peptides and may explain the finding that GNGRG peptide can target TNF to tumors, albeit to a lower extent than CNGRC. The disulfide bridge constraint is critical for stabilizing the bent conformation and for increasing the tumor targeting efficiency.     

Study of the insulin dimerization: binding free energy calculations and per-residue free energy decomposition

A calculation of the binding free energy for the dimerization of insulin has been performed using the molecular mechanics-generalized Born surface area approach. The calculated absolute binding free energy is -11.9 kcal/mol, in approximate agreement with the experimental value of -7.2 kcal/mol. The results show that the dimerization is mainly due to nonpolar interactions. The role of the hydrogen bonds between the 2 monomers appears to give the direction of the interactions. A per-atom decomposition of the binding free energy has been performed to identify the residues contributing most to the self association free energy. Residues B24-B26 are found to make the largest favorable contributions to the dimerization. Other residues situated at the interface between the 2 monomers were found to make favorable but smaller contributions to the dimerization: Tyr B16, Val B12, and Pro B28, and to an even lesser extent, Gly B23. The energy decomposition on a per-residue basis is in agreement with experimental alanine scanning data. The results obtained from a single trajectory (i.e., the dimer trajectory is also used for the monomer analysis) and 2 trajectories (i.e., separate trajectories are used for the monomer and dimer) are similar.     

Structure-activity relationships in carbohydrates revealed by their hydration

One of the more intriguing aspects of carbohydrate chemistry is that despite having very similar molecular structures, sugars have very different properties. For instance, there is a sensible difference in sweet taste between glucose and trehalose, even though trehalose is a disaccharide that comprised two glucose units, suggesting a different ability of these two carbohydrates to bind to sweet receptors. Here we have looked at the hydration of specific sites and at the three-dimensional configuration of water molecules around three carbohydrates (glucose, cellobiose, and trehalose), combining neutron diffraction data with computer modelling. Results indicate that identical chemical groups can have radically different hydration patterns depending on their location on a given molecule. These differences can be linked with the specific activity of glucose, cellobiose, and trehalose as a sweet substance, as building block of cellulose fiber, and as a bioprotective agent, respectively. This article is part of a Special Issue entitled “Recent Advances in Bionanomaterials” Guest Editors: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.     

Conformation-specific affinity purification of proteins using engineered binding proteins: application to the estrogen receptor

Affinity chromatography coupled with an "affinity tag" has become a powerful and routine technology for the purification of recombinant proteins. However, such tag-based affinity chromatography usually cannot separate different conformational states (e.g., folded and misfolded) of a protein to be purified. Here, we describe a strategy to separate different conformations of a protein by using "tailor-made" affinity chromatography based on engineered binding proteins. Our method involves: (i) engineering of a binding protein specific to a particular conformation of the protein of interest, and (ii) production and immobilization of the binding protein to prepare conformation-specific affinity chromatography media. Using "monobodies," small antibody mimics based on the fibronectin type III domain, as the target-binding proteins, we demonstrated the effectiveness of our method by separating the active form of the estrogen receptor alpha ligand-binding domain (ERalpha-LBD) from a mixture of active and misfolded species and by discriminating two different conformations of ERalpha-LBD bound to different ligands. Our strategy should be generally applicable to the preparation of conformationally homogeneous protein samples.     

Free energy of proton binding in proteins

In this article we use literature data on the titration of denatured ribonuclease to test the accuracy of proton-binding distributions obtained using our recent approach employing moments. We find that using only the local slope of the titration curve at a small number of points (five, for example) we can reproduce the detailed proton-binding distribution at all pH values. Our method gives the complete proton-binding polynomial for a given protein and each coefficient in this polynomial in turn yields the free energy for binding a given number of protons in all ways to the protein. Using these net free energies, we can then compute the average proton-binding free energy per proton as a function of the fraction of protons bound. We find that this function is remarkably similar for different proteins, even for proteins that exhibit quite different titration behavior. For the special case of binding to independent sites, we obtain simple relations for the first and last terms in the free energy per-proton function. For this special case we also can calculate the distribution functions giving the probability that a molecule has a given number of positive or negative charges and the joint distribution that a molecule simultaneously has a given number of positive and negative charge.