Investigations of neurotrophic inhibitors of FK506 binding protein via Monte Carlo simulations

The binding and solution-phase properties of six inhibitors of FK506 binding protein (FKBP12) were investigated using free energy perturbation techniques in Monte Carlo statistical mechanics simulations. These nonimmunosuppressive molecules are of current interest for their neurotrophic activity when bound to FKBP12 as well as for their potential as building blocks for chemical inducers of protein dimerization. Relative binding affinities were computed and analyzed for ligands differing by a phenyl ring, an external phenyl or pyridyl substituent, and a pipecolyl or prolyl ring. Such results are, in general, valuable for inhibitor optimization and, in the present case, bring into question some of the previously reported binding data.     

Conformations of peptide fragments from the FK506 binding protein: comparison with the native and urea-unfolded states

The helix-forming tendency of seven peptide fragments corresponding with the entire sequence of the FK506 binding protein (FKBP) has been investigated in aqueous buffer and in 2,2,2-trifluoroethanol (TFE) using CD and NMR spectroscopy. All fragments exhibited random coil conformations in aqueous buffer, whereas the amount of helix induced in the peptide fragments by TFE varied. The fragment with the highest degree of helicity in TFE corresponded with the single (alpha-helix in native FKBP. Fragments corresponding with beta-strands 2 and 3 also exhibited strong propensity towards helix formation. In contrast, the fragment corresponding with beta-strand 1 did not form helix in TFE. The inherent helix-forming tendencies are interpreted in light of the native structure to suggest possible folding nucleation sites. Conformational sampling in each peptide fragment was also compared with that observed in urea-denatured FKBP. With the exception of the fragment corresponding with beta-strand 2, the formation of helical structures in the peptide fragments in TFE was correlated with the observation of turn and/or helix conformers in urea-unfolded FKBP. Surprisingly, peptide fragments in aqueous solution were less structured than the corresponding regions in urea-denatured FKBP. The conformational differences between the peptide fragments and unfolded FKBP were not due to the urea buffer or to differences in their rotational correlation times. We conclude that local amino acid interactions are not generally sufficient to account for the formation of non-random conformations in unfolded FKBP. Formation of non-random structures in unfolded FKBP may require stabilization of incipient turn or helical conformations through transient contact with non-local non-polar residues.     

Modeling the interaction between FK506 and FKBP12: a mechanism for formation of the calcineurin inhibitory complex

FK506 is a naturally occurring immunosuppressant whose mode of action involves formation of an initial complex with the cytosolic protein FKBP12. The composite surface of this complex then binds to and inhibits the protein phosphatase calcineurin (PP2B). To investigate why FK506 does not inhibit calcineurin directly we have conducted molecular modeling and conformational studies on published structures of FK506 both alone and in complex with FKBP12. From studies of the structure of FK506 in CDCl3 and Z-Arg32-ascomycin in water (a water soluble analogue of FK506) we suggest that the FK506 molecule can be viewed as consisting of three separate regions. The pipecolate region which extends from C24 to C10 including the pipecolate ring shows strongly conserved conformation in both solvents. The loop region which extends from C25 to C16 shows general conservation of the loop structure and the pyranose region made up of the pyranose ring and C15-C17 which shows highly variable conformation depending on solvent. Comparison of the structure of Z-Arg32-ascomycin in water with structures of FK506 bound to FKBP12 indicate that the conformation of the pipecolate region is conserved during the binding process. The conformation of the loop region was generally conserved but a significant reduction (approximately 1.7 A) in the diameter of the loop in the bound structure was observed. The conformation of the pyranose ring and C15-C17 region was found to be significantly altered in the bound structure resulting in displacements of the C13 and C15 methoxyl groups of 2.8 and 3.5 A, respectively. From computer models and molecular dynamics simulations of interactions between FK506 and FKBP12 we suggest that the conformational changes observed in bound FK506 are induced by the interaction between the 80's loop of FKBP12 and the pyranose ring of FKBP12. These interactions result in the formation of a complex with the both correct shape and surface polarity for interaction with calcineurin.     

Locations of calmodulin and FK506-binding protein on the three-dimensional architecture of the skeletal muscle ryanodine receptor

Isolated skeletal muscle ryanodine receptors (RyRs) complexed with the modulatory ligands, calmodulin (CaM) or 12-kDa FK506-binding protein (FKBP12), have been characterized by electron cryomicroscopy and three-dimensional reconstruction. RyRs are composed of 4 large subunits (molecular mass 565 kDa) that assemble to form a 4-fold symmetric complex that, architecturally, comprises two major substructures, a large ( approximately 80% of the total mass) cytoplasmic assembly and a smaller transmembrane assembly. Both CaM and FKBP12 bind to the cytoplasmic assembly at sites that are 10 and 12 nm, respectively, from the putative entrance to the transmembrane ion channel. FKBP12 binds along the edge of the square-shaped cytoplasmic assembly near the face that interacts in vivo with the sarcolemma/transverse tubule membrane system, whereas CaM binds within a cleft that faces the junctional face of the sarcoplasmic reticulum membrane at the triad junction. Both ligands interact with a domain that connects directly to a cytoplasmic extension of the transmembrane assembly of the receptor, and thus might cause structural changes in the domain which in turn modulate channel gating.     

Site-directed mutational analysis for the membrane binding of DnaA protein. Identification of amino acids involved in the functional interaction between DnaA protein and acidic phospholipids

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, interacts with acidic phospholipids, such as cardiolipin, and its activity seems to be regulated by membrane binding in cells. In this study we introduced site-directed mutations at the positions of hydrophobic or basic amino acids which are conserved among various bacteria species and which are located in the putative membrane-binding region of DnaA protein (from Asp357 to Val374). All mutant DnaA proteins showed much the same ATP and ADP binding activity as that of the wild-type protein. The release of ATP bound to the mutant DnaA protein, in which three hydrophobic amino acids were mutated to hydrophilic ones, was stimulated by cardiolipin, as in the case of the wild-type protein. On the other hand, the release of ATP bound to another mutant DnaA protein, in which three basic amino acids were mutated to acidic ones, was not stimulated by cardiolipin. These results suggest not only that the region is a membrane-binding domain of DnaA protein but also that these basic amino acids are important for the binding and the ionic interaction between the basic amino acids and acidic residues of cardiolipin and is involved in the interaction between DnaA protein and cardiolipin.     

Relationship of whole-blood FK506 concentrations to rejection and toxicity in liver and kidney transplants

FK506 (tacrolimus) has been shown to be a safe and effective immunosuppressant for the prevention of organ rejection after liver and kidney transplantation. Like cyclosporine, the use of FK506 has been associated with some adverse effects such as toxicity and organ rejection. Therapeutic monitoring of the whole-blood FK506 drug concentrations has been used in an effort to determine how the concentration of FK506 in the blood is related to the development of toxicity or the risk for organ rejection. Cox regression analysis of two recent clinical trials of FK506 in patients receiving kidney and liver transplants shows a significant correlation between the whole-blood FK506 concentrations and the incidence of both toxicity and organ rejection. Because of these relationships and the pharmacokinetics of FK506, therapeutic monitoring of the whole-blood FK506 levels is expected to be helpful for minimizing the risks of both toxicity and rejection in liver and kidney transplants.     

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

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

Mutational analysis of the major loop of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases. Effects on protein stability and substrate binding

The carbohydrate-binding cleft of Bacillus licheniformis 1,3-1, 4-beta-D-glucan 4-glucanohydrolase is partially covered by the surface loop between residues 51 and 67, which is linked to beta-strand-(87-95) of the minor beta-sheet III of the protein core by a single disulfide bond at Cys61-Cys90. An alanine scanning mutagenesis approach has been applied to analyze the role of loop residues from Asp51 to Arg64 in substrate binding and stability by means of equilibrium urea denaturation, enzyme thermotolerance, and kinetics. The DeltaDeltaGU between oxidized and reduced forms is approximately constant for all mutants, with a contribution of 5.3 +/- 0.2 kcal.mol-1 for the disulfide bridge to protein stability. A good correlation is observed between DeltaGU values by reversible unfolding and enzyme thermotolerance. The N57A mutant, however, is more thermotolerant than the wild-type enzyme, whereas it is slightly less stable to reversible urea denaturation. Mutants with a <2-fold increase in Km correspond to mutations at residues not involved in substrate binding, for which the reduction in catalytic efficiency (kcat/Km) is proportional to the loss of stability relative to the wild-type enzyme. Y53A, N55A, F59A, and W63A, on the other hand, show a pronounced effect on catalytic efficiency, with Km > 2-fold and kcat < 5% of the wild-type values. These mutated residues are directly involved in substrate binding or in hydrophobic packing of the loop. Interestingly, the mutation M58A yields an enzyme that is more active than the wild-type enzyme (7-fold increase in kcat), but it is slightly less stable.     

The contributions of individual gamma-carboxyglutamic acid residues in the calcium-dependent binding of recombinant human protein C to acidic phospholipid vesicles

The dependence on the integrity of the human protein C (PC) gamma-carboxyglutamic acid (Gla) domain on its Ca(2+)-dependent binding properties to acidic phospholipid (PL) vesicles has been examined by analysis of these interactions with recombinant (r)-PC Gla domain muteins. The concentration of Ca2+ that results in 50% saturation (C50-Ca) of PL with wild-type (wt) r-PC was not altered by more than 2-fold for the following r-variants of PC, viz. [Gla6-->Asp]r-PC, [Gla14-->Asp]r-PC, [Gla19-->Asp]r-PC, [Gla25-->Asp]r-PC, [Gla29-->Asp]r-PC, and [Gln32-->Gla]r-PC. The C50-Ca was substantially higher than that of wtr-PC for [Gla7-->Asp]r-PC (8.2 mM), [Arg15-->Leu]r-PC (4.5 mM), [Gla16-->Asp]r-PC (> 10 mM), [Gla20-->Asp]r-PC (> 10 mM), and [Gla26-->Asp]r-PC (> 10 mM), indicating that the Ca(2+)-induced conformations of these latter variants interacted poorly with these acidic PL vesicles. Titration of the PL vesicles with wtr-PC at a constant concentration of 20 mM Ca2+ leads to calculation of a concentration of PC that results in 50% saturation of the PL (C50-PC) of 0.38 microM. Essentially this same value was determined for the r-mutants, [Gla7-->Asp]r-PC and [Gln32-->Gla]r-PC. An approximate 2-fold lower C50-PC was obtained for [Gla14-->Asp]r-PC (0.14 microM), [Gla25-->Asp]r-PC (0.16 microM), and [Gla29-->Asp]r-PC (0.15 microM). Somewhat higher C50-PC values were found for [Gla6-->Asp]r-PC (1.2 microM), [Arg15-->Leu]r-PC (1.2 microM), [Gla16-->Asp]r-PC (1.2 microM), [Gla19-->Asp]r-PC (1.8 microM), [Gla20-->Asp]r-PC (1.1 microM), and [Gla26-->Asp]r-PC (1.6 microM). The results of this investigation, in conjunction with other structural and functional studies with these mutants, and the x-ray crystallographic structure of the prothrombin Gla domain-Ca2+ complex, show that Gla16 and Gla26 are the most indispensable Gla residues for maintenance of the functional Ca(2+)-dependent structure of the Gla domain of PC, whereas Gla14 is the least critical Gla residue in this regard. Of the non-Gla residues investigated, Arg15 is of great importance for maintenance of a functional Ca(2+)-dependent structure of PC, and insertion of a Gla residue at position 32, a situation that exists in the cases of some other proteins of this type, does not significantly alter these characteristics of r-PC.     

Electrostatic and non-electrostatic contributions to the binding free energies of anthracycline antibiotics to DNA

The knowledge about molecular factors driving simple ligand-DNA interactions is still limited. The aim of the present study was to investigate the electrostatic and non-electrostatic contributions to the binding free energies of anthracycline compounds with DNA. Theoretical calculations based on continuum methods (Poisson-Boltzmann and solvent accessible surface area) were performed to estimate the binding free energies of five selected anthracycline ligands (daunomycin, adriamycin, 9-deoxyadriamycin, hydroxyrubicin, and adriamycinone) to DNA. The free energy calculations also took into account the conformational change that DNA undergoes upon ligand binding. This conformational change appeared to be very important for estimating absolute free energies of binding. Our studies revealed that the absolute values of all computed contributions to the binding free energy were quite large compared to the total free energy of binding. However, the sum of these large positive and negative values produced a small negative value of the free energy around -10 kcal/mol. This value is in good agreement with experimental data. Experimental values for relative binding free energies were also reproduced for charged ligands by our calculations. Together, it was found that the driving force for ligand-DNA complex formation is the non-polar interaction between the ligand and DNA even if the ligand is positively charged.     

Social psychological contributions to the decade of the brain: Doctrine of multilevel analysis.

The 1990s were declared by Congress to be the "decade of the brain." This declaration is important to all psychologists, not only neuroscientists, because with this declaration come expectations of the cognitive and behavioral sciences generally and because the brain does not exist in isolation but rather is a fundamental component of developing and aging individuals who themselves are mere actors in the larger theater of life. This article examines the importance of a multilevel, integrative approach to the study of mental and behavioral phenomena in the decade of the brain, reviews how this approach highlights the synergistic relationship between theoretical and clinically relevant research, and illustrates how this approach can foster the transition from microtheories to general psychological theories. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Dynamic contributions to the DNA binding entropy of the EcoRI and EcoRV restriction endonucleases

Molecular Dynamics simulations on DNA-EcoRI and DNA-EcoRV complexes suggest that the DNA within these complexes is significantly more ordered than free DNA. Similarly, both the protein and the DNA are more ordered in the specific (cognate) DNA-EcoRV complex than they are in the non-cognate DNA-protein complex, consistent with recently proposed analogies between protein folding and sequence-specific DNA-protein recognition. Analysis of the trajectories shows that the net entropy gain upon specific binding to be the result of opposing contributions. Solvent release, which increases entropy versus configurational terms (as measured by the magnitude of the atomic fluctuations), and collective terms from tight coupling between the motions of the protein and the DNA.     

Site-directed mutational analysis for the ATP binding of DnaA protein. Functions of two conserved amino acids (Lys-178 and Asp-235) located in the ATP-binding domain of DnaA protein in vitro and in vivo

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, is activated by binding to ATP in vitro. We introduced site-directed mutations into two amino acids of the protein conserved among various ATP-binding proteins and examined functions of the mutated DnaA proteins, in vitro and in vivo. Both mutated DnaA proteins (Lys-178 --> Ile or Asp-235 --> Asn) lost the affinity for both ATP and ADP but did maintain binding activity for oriC. Specific activities in an oriC DNA replication system in vitro were less than one-tenth those of the wild-type protein. Assay of the generation of oriC sites sensitive to P1 nuclease, using the mutated DnaA proteins, revealed a defect in induction of the duplex opening at oriC. On the other hand, expression of each mutated DnaA protein in the temperature-sensitive dnaA46 mutant did not complement the temperature sensitivity. We suggest that Lys-178 and Asp-235 of DnaA protein are essential for the activity needed to initiate oriC DNA replication in vitro and in vivo and that ATP binding to DnaA protein is required for DNA replication-related functions.     

Computer-based analysis of the binding steps in protein complex formation

Computer models were used to examine whether and under what conditions the multimeric protein complex is inhibited by high concentrations of one of its components-an effect analogous to the prozone phenomenon in precipitin tests. A series of idealized simple "ball-and-stick" structures representing small oligomeric complexes of protein molecules formed by reversible binding reactions were analyzed to determine the binding steps leading to each structure. The equilibrium state of each system was then determined over a range of starting concentrations and Kds and the steady-state concentration of structurally complete oligomer calculated for each situation. A strong inhibitory effect at high concentrations was shown by any protein molecule forming a bridge between two or more separable parts of the complex. By contrast, proteins linked to the outside of the complex by a single bond showed no inhibition whatsoever at any concentration. Nonbridging, multivalent proteins in the body of the complex could show an inhibitory effect or not depending on the structure of the complex and the strength of its bonds. On the basis of this study, we suggest that the prozone phenomenon will occur widely in living cells and that it could be a crucial factor in the regulation of protein complex formation.     

The carbohydrate recognition domain of surfactant protein A mediates binding to the major surface glycoprotein of Pneumocystis carinii

Pneumocystis carinii is a common cause of life-threatening pneumonia in immunodeficient patients. Pulmonary surfactant protein A (SP-A), an alveolar glycoprotein containing collagen-like and carbohydrate recognition domains (CRD), binds P. carinii and enhances adherence to alveolar macrophages. In this study, we examined the structural basis of the interaction between SP-A and the major surface glycoprotein of P. carinii (MSG). Rat SP-A bound to purified rat P. carinii-derived MSG in a saturable and calcium-dependent manner, which was partially reversible by coincubation with excess monosaccharides, or pretreatment of MSG with N-glycanase. Mutant recombinant SP-As with neutral amino acid substitutions for the predicted calcium- and carbohydrate-coordinating residues of the CRD were synthesized in insect cells using baculovirus vectors and tested for binding to MSG. Substitutions of negatively charged (Glu195, Glu202, and Asp215) and polar residues (Asn214) of the CRD with alanine but not substitution of the Arg197 with glycine reduced the binding of SP-A to mannose-Sepharose beads and to MSG. Deletion of the N-linked oligosaccharides from SP-A by mutagenesis of the consensus sequences for glycosylation had no effect on binding. We conclude that the CRD mediates the binding of SP-A to oligosaccharides attached to MSG.     

Defining the arachidonic acid binding site of human 15-lipoxygenase. Molecular modeling and mutagenesis

Mammalian lipoxygenases have been implicated in the pathogenesis of several inflammatory disorders and are, therefore, important targets for drug discovery. Both plant and mammalian lipoxygenases catalyze the dioxygenation of polyunsaturated fatty acids, which contain one or more 1,4-cis,cis-pentadiene units to yield hydroperoxide products. At the time this study was initiated, soybean lipoxygenase-1 was the only lipoxygenase for which an atomic resolution structure had been determined. No structure of lipoxygenase with substrate or inhibitor bound is currently available. A model of arachidonic acid docked into the proposed substrate binding site in the soybean structure is presented here. Analysis of this model suggested two residues, an aromatic residue and a positively charged residue, could be critical for substrate binding. Validation of this model is provided by site-directed mutagenesis of human 15-lipoxygenase, despite the low amino acid sequence identity between the soybean and mammalian enzymes. Both a positively charged amino acid residue (Arg402) and an aromatic amino acid residue (Phe414) are identified as critical for the binding of fatty acid substrates in human 15-lipoxygenase. Thus, binding determinants shown to be characteristic of non-enzymatic fatty acid-binding proteins are now implicated in the substrate binding pocket of lipoxygenases.