Evidence for the Preservation of Native Inter- and Intra-Molecular Hydrogen Bonds in the Desolvated FK-Binding Protein·FK506 Complex Produced by Electrospray Ionization

It is now well established that electrospray ionization (ESI) is capable of introducing noncovalent protein assemblies into a desolvated environment, thereby allowing their analysis by mass spectrometry. The degree to which native interactions from the solution phase are preserved in this environment is less clear. Site-directed mutagenesis of FK506-binding protein (FKBP) has been employed to probe specific intra- and inter-molecular interactions within the complex between FKBP and its ligand FK506. Collisional activation of wild-type and mutant-FKBP?FK506 ions, generated by ESI, demonstrated that removal of native protein-ligand interactions formed between residues Asp37, Tyr82, and FK506 significantly destabilized the complex. Mutation of Arg42 to Ala42, or Tyr26 to Phe26 also resulted in lower energy dissociation of the FKBP·FK506 complex. Although these residues do not form direct H-bonds to FK506, they interact with Asp37, ensuring its correct orientation to associate with the ligand. Comparison with solution-based affinity measurements of these mutants has been discussed, including the stabilization afforded by ordered water molecules. Ion mobility spectrometry (IMS) has been employed to provide gas-phase structural information on the unfolding of the complexes. The [M + 6H](6+) complexes of the wild-type and mutants have been shown to resist unfolding and retain compact conformations. However, removal of the basic Arg42 residue was found to induce significant structural weakening of the [M + 7H](7+) complex when raised to dissociation-level energies. Overall, destabilization of the FKBP·FK506 complex, resulting from targeted removal of specific H-bonds, provides evidence for the preservation of these interactions in the desolvated wild-type complex.     

Detection of noncovalent FKBP-FK506 and FKBP-Rapamycin complexes by capillary electrophoresis-mass spectrometry and capillary electrophoresis-tandem mass spectrometry

The well known biospecific noncovalent receptor-ligand association complexes between the immunophilin FKBP and the immunosuppressive drugs FK506 and Rapamycin (RM) were investigated by on-line capillary electrophoresis-mass spectrometry (CE-MS) under selected ion monitoring (SIM) conditions and by CE-MS with tandem mass spectrometry (CE-MS/MS) under selected reaction monitoring (SRM) conditions. Solutions of hFKBP (33.3 µM) were dissolved in 50 mM ammonium acetate at pH 7.5. Samples that contained 100 µM of FK506 or RM also were prepared under the same solution conditions. By using these aqueous pH neutral conditions, samples were analyzed by SIM CE-MS and SRM CE-MS and the target complexes were separated by CE with mass spectrometer detection of the individual complexes between FKBP and FK506 [hFKBP + FK506 + 7HJ⁷⁺ as well as FKBP and RM [hFKBP + RM + 7HJ⁷⁺. In an experiment where a mixture of FK506 and RM was analyzed in the presence of FKBP, a nine-to-one ratio of ion current abundances between the RM and FK506 complexes was observed as reported in the literature from other studies. These results suggest that CE-MS and CE-MS/MS may be yet another analytical method for studying noncovalent interactions of biologically important macromolecules under physiological conditions.     

Study of the effect of Wuzhi tablet (Schisandra sphenanthera extract) on tacrolimus tissue distribution in rat by liquid chromatography tandem mass spectrometry method

A liquid chromatography/tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determining tacrolimus (FK506) in rat tissues to study the effect of Schisandra sphenanthera extract on FK506 tissue distribution. After a liquid–liquid extraction with ethyl acetate, FK506 and ascomycin (IS) were subjected to LC‐MS/MS analysis using positive electrospray ionization under multiple reactions monitoring mode. Chromatographic separation of FK506 and ascomycin was achieved on a Hypersil BDS C₁₈ column with a mobile phase consisting of methanol‐water (containing 2 mM ammonium acetate, 95 : 5, v/v). The intra‐ and inter‐batch precision of the method were less than 8.8 and 9.8%, respectively. The intra‐ and inter‐batch accuracies ranged from 97.5 to 104.0%. The lowest limit of quantification for FK506 was 0.5 ng/mL. The method was applied to a FK506 tissue distribution study with or without a dose of Wuzhi (WZ) tablet. Most of the FK506 tissue concentrations were slightly increased after a concomitant WZ tablet dose, but the whole blood concentration of FK506 was dramatically increased 3‐fold after a concomitant WZ tablet dose. These results indicated that the LC‐MS/MS method was rapid and sensitive enough to quantify FK506 in different rat tissues, and strict drug monitoring is recommended when co‐administering WZ tablet in clinical use. Copyright © 2009 John Wiley & Sons, Ltd.     

Involvement of some large immunophilins and their ligands in the protection and regeneration of neurons: a hypothetical mode of action

The powerful immunosuppressive drugs such as FK506 and its derivatives induce some regeneration and protection of neurons from ischaemic brain injury and some other neurological disorders. The drugs form complexes with diverse FKBPs but apparently the FKBP52/FK506 complex was shown to be involved in the protection and regeneration of neurons. We used several different sequence attributes in searching diverse genomic databases for similar motifs as those present in the FKBPs. A Fortran library of algorithms (Par_Seq) has been designed and used in searching for the similarity of sequence motifs extracted from the multiple sequence alignments of diverse groups of proteins (query motifs) and the target motifs which are encoded in various genomes. The following sequence attributes were used in the establishment of the degree of convergence between: (A) amino acid (AA) sequence similarity (ID) of the query/target motifs and (B) their: (1) AA composition (AAC); (2) hydrophobicity (HI); (3) Jensen-Shannon entropy; and (4) AA propensity to form a particular secondary structure. The sequence hallmark of two different groups of peptidylprolyl cis/trans isomerases (PPIases), namely tetratricopetide repeat (TPR) motifs, which are present in the heat-shock cyclophilins and in the large FK506-binding proteins (FKBPs) were used to search various genomic databases. The Par_Seq algorithm has revealed that the TPR motifs have similar sequence attributes as a number of hydrophobic sequence segments of functionally unrelated membrane proteins, including some of the TMs from diverse G protein-coupled receptors (GPCRs). It is proposed that binding of the FKBP52/FK506 complex to the membranes via the TPR motifs and its interaction with some membrane proteins could be in part responsible for some neuro-regeneration and neuro-protection of the brain during some ischaemia-induced stresses.     

Development of 7-membered N-heterocyclic carbene ligands for transition metals

We recently reported the first example of a seven-membered N-heterocyclic carbene (NHC) ligand for transition metals. These ligands are attractive because the heterocyclic framework, derived from 2,2′-diaminobiphenyl, exhibits a torsional twist that results in a chiral, C₂-symmetric structure. The present report outlines the synthetic efforts that led to the development of these ligands together with the synthesis and structural characterization of metal complexes bearing seven-membered NHCs as ancillary ligands. The identity of nitrogen substituent, neopentyl versus 2-adamantyl, influences the synthetic accessibility and stability of the seven-membered amidinium salts and the NHC–metal complexes obtained via in situ deprotonation/metallation. Computational analysis of the seven-membered ring structures reveals the Hückel antiaromatic 8π electron system achieves significant Möbius aromatic stabilization upon undergoing torsional distortion of the heterocyclic ring.     

Conformational Transitions and Convergence of Absolute Binding Free Energy Calculations

The Binding Energy Distribution Analysis Method (BEDAM) is employed to compute the standard binding free energies of a series of ligands to a FK506 binding protein (FKBP12) with implicit solvation. Binding free energy estimates are in reasonably good agreement with experimental affinities. The conformations of the complexes identified by the simulations are in good agreement with crystallographic data, which was not used to restrain ligand orientations. The BEDAM method is based on λ -hopping Hamiltonian parallel Replica Exchange (HREM) molecular dynamics conformational sampling, the OPLS-AA/AGBNP2 effective potential, and multi-state free energy estimators (MBAR). Achieving converged and accurate results depends on all of these elements of the calculation. Convergence of the binding free energy is tied to the level of convergence of binding energy distributions at critical intermediate states where bound and unbound states are at equilibrium, and where the rate of binding/unbinding conformational transitions is maximal. This finding mirrors similar observations in the context of order/disorder transitions as for example in protein folding. Insights concerning the physical mechanism of ligand binding and unbinding are obtained. Convergence for the largest FK506 ligand is achieved only after imposing strict conformational restraints, which however require accurate prior structural knowledge of the structure of the complex. The analytical AGBNP2 model is found to underestimate the magnitude of the hydrophobic driving force towards binding in these systems characterized by loosely packed protein-ligand binding interfaces. Rescoring of the binding energies using a numerical surface area model corrects this deficiency. This study illustrates the complex interplay between energy models, exploration of conformational space, and free energy estimators needed to obtain robust estimates from binding free energy calculations.     

Parameterization of OPLS-AA force field for the conformational analysis of macrocyclic polyketides

The parameters for the OPLS-AA potential energy function have been extended to include some functional groups that are present in macrocyclic polyketides. Existing OPLS-AA torsional parameters for alkanes, alcohols, ethers, hemiacetals, esters, and ketoamides were improved based on MP2/aug-cc-pVTZ and MP2/aug-cc-pVDZ calculations. Nonbonded parameters for the sp(3) carbon and oxygen atoms were refined using Monte Carlo simulations of bulk liquids. The resulting force field predicts conformer energies and torsional barriers of alkanes, alcohols, ethers, and hemiacetals with an overall RMS deviation of 0.40 kcal/mol as compared to reference data. Densities of 19 bulk liquids are predicted with an average error of 1.1%, and heats of vaporization are reproduced within 2.4% of experimental values. The force field was used to perform conformational analysis of smaller analogs of the macrocyclic polyketide drug FK506. Structures that adopted low-energy conformations similar to that of bound FK506 were identified. The results show that a linker of four ketide units constitutes the shortest effector domain that allows binding of the ketide drugs to FKBP proteins. It is proposed that the exact chemical makeup of the effector domain has little influence on the conformational preference of tetraketides.     

Pose scoring by NMR

Recently, we have developed a fast approach to calculate NMR chemical shifts using the divide and conquer method at the semiempirical level. To demonstrate the utility of this approach for characterizing protein-ligand interactions, we used the deviation of calculated chemical shift perturbations from experiment to determine the orientation of a ligand (GPI-1046) in the binding pocket of the FK506 binding protein (FKBP12). Moreover, we were able to select the native state of the ligand from a collection of decoy poses. A key hydrogen bond between O1 and HN in Ile56 was also identified. Our results suggest that ligand-induced chemical shift perturbations can be used to refine protein/ligand structures.     

The Crystal Structure of the Macrocyclic Hexathioether 1,4,7,11,14,17-Hexathiacycloeicosane: Implications for Metal Complexation

Several metal complexes of the twenty-membered ring hexathioether macrocycle, 1,4,7,11,14,17-hexathiacycloeicosane (20S6), have now been prepared and crystallographically characterized. In order to examine structural changes in the ligand which may occur under complexation, we undertook an analysis of the structure of the 20S6 ligand by single crystal X-ray diffraction. The compound crystallizes in the monoclinic space group C₂/c with the unit cell dimensions: a = 22.481(1) Å, b = 5.433(2) Å, c = 17.9259(9) Å, and = 117.711(4)°. The conformation adopted by this ligand is such that four of the sulfur atoms are exodentate, but two sulfur atoms are syn endodentate, unusual for a macrocycle of this type. All twelve of the C—S—C—C torsional bond angles in 20S6 are gauche as expected, and the ligand conformation may account for the complexation properties observed for this hexathioether macrocycle.     

Native FKBP12 engineering by ligand-directed tosyl chemistry: labeling properties and application to photo-cross-linking of protein complexes in vitro and in living cells

The ability to modify target "native" (endogenous) proteins selectively in living cells with synthetic molecules should provide powerful tools for chemical biology. To this end, we recently developed a novel protein labeling technique termed ligand-directed tosyl (LDT) chemistry. This method uses labeling reagents in which a protein ligand and a synthetic probe are connected by a tosylate ester group. We previously demonstrated its applicability to the selective chemical labeling of several native proteins in living cells and mice. However, many fundamental features of this chemistry remain to be studied. In this work, we investigated the relationship between the LDT reagent structure and labeling properties by using native FK506-binding protein 12 (FKBP12) as a target protein. In vitro experiments revealed that the length and rigidity of the spacer structure linking the protein ligand and the tosylate group have significant effects on the overall labeling yield and labeling site. In addition to histidine, which we reported previously, tyrosine and glutamate residues were identified as amino acids that are modified by LDT-mediated labeling. Through the screening of various spacer structures, piperazine was found to be optimal for FKBP12 labeling in terms of labeling efficiency and site specificity. Using a piperazine-based LDT reagent containing a photoreactive probe, we successfully demonstrated the labeling and UV-induced covalent cross-linking of FKBP12 and its interacting proteins in vitro and in living cells. This study not only furthers our understanding of the basic reaction properties of LDT chemistry but also extends the applicability of this method to the investigation of biological processes in mammalian cells.     

Nocardiopsins: New FKBP12‐Binding Macrolide Polyketides from an Australian Marine‐Derived Actinomycete,Nocardiopsis sp.

A marine‐derived actinomycete, Nocardiopsis sp. (CMB‐M0232), obtained from a sediment sample collected at a depth of 55 m off the coast of Brisbane, Australia, yielded two new macrolide polyketides. Structures for nocardiopsins A and B were assigned by detailed spectroscopic analysis, degradation and chemical derivatization. A Marfey’s analysis revealed an unexpected acid‐mediated partial racemization of the L ‐pipecolic acid incorporated within the nocardiopsins. The scope of this racemization was assessed against a selection of natural and synthetic N‐acyl pipecolic acids. While the nocardiopsins are not antibacterial, antifungal or cytotoxic, they do exhibit low‐micromolar binding to the immunophilin FKBP12, consistent with their structural and biosynthetic relationship to the immunosuppressive agents FK506 and rapamycin. The nocardiopsins represent a new point of entry into what has been a valuable, exclusive and reclusive region of bioactive chemical space—that surrounding the FK506/rapamycin pharmacophore.     

Chemical genetic control of protein levels: selective in vivo targeted degradation

Genetic loss of function analysis is a powerful method for the study of protein function. However, some cell biological questions are difficult to address using traditional genetic strategies often due to the lack of appropriate genetic model systems. Here, we present a general strategy for the design and syntheses of molecules capable of inducing the degradation of selected proteins in vivo via the ubiquitin-proteasome pathway. Western blot and fluorometric analyses indicated the loss of two different targets: green fluorescent protein (GFP) fused with FK506 binding protein (FKBP12) and GFP fused with the androgen receptor (AR), after treatment with PROteolysis TArgeting Chimeric moleculeS (PROTACS) incorporating a FKBP12 ligand and dihydrotestosterone, respectively. These are the first in vivo examples of direct small molecule-induced recruitment of target proteins to the proteasome for degradation upon addition to cultured cells. Moreover, PROTAC-mediated protein degradation offers a general strategy to create "chemical knockouts," thus opening new possibilities for the control of protein function.     

Strongly Absorbing π–π* States in Heteroleptic Dipyrrin/2,2′‐Bipyridine Ruthenium Complexes: Excited‐State Dynamics from Resonance Raman Spectroscopy

A recently reported new class of ruthenium complexes containing 2,2′‐bipyridine and a dipyrrin ligand in the coordination sphere exhibit both strong metal‐to‐ligand charge‐transfer (MLCT) and π–π* transitions. Quantitative analysis of the resonance Raman scattering intensities and absorption spectra reveals only weak electronic interactions between these states despite direct coordination of the bipyridyl and dipyrrin ligands to the central ruthenium atom. On the basis of DFT calculations and time‐dependent DFT (TD‐DFT), we propose that the electronic excited states closely resemble “pure” MLCT and π–π* states. Resonance Raman intensity analysis demonstrates that a large amplitude transannular torsional motion provides a mechanism for relaxation on the π–π* excited‐state surface. We assert that this result is generally applicable to a range of dipyrrin complexes such as boron–dipyrrin and metallodipyrrin systems. Despite the large torsional distortion between the phenyl ring and the dipyrromethene plane, π–π* excitation extends out onto the phenyl ring which may have important consequences in solar‐energy‐conversion applications of ruthenium–dipyrrin complexes.     

Connectivity and binding‐site recognition: Applications relevant to drug design

Here, we describe a family of methods based on residue–residue connectivity for characterizing binding sites and apply variants of the method to various types of protein–ligand complexes including proteases, allosteric‐binding sites, correctly and incorrectly docked poses, and inhibitors of protein–protein interactions. Residues within ligand‐binding sites have about 25% more contact neighbors than surface residues in general; high‐connectivity residues are found in contact with the ligand in 84% of all complexes studied. In addition, a k‐means algorithm was developed that may be useful for identifying potential binding sites with no obvious geometric or connectivity features. The analysis was primarily carried out on 61 protein–ligand structures from the MEROPS protease database, 250 protein–ligand structures from the PDBSelect (25%), and 30 protein–protein complexes. Analysis of four proteases with crystal structures for multiple bound ligands has shown that residues with high connectivity tend to have less variable side‐chain conformation. The relevance to drug design is discussed in terms of identifying allosteric‐binding sites, distinguishing between alternative docked poses and designing protein interface inhibitors. Taken together, this data indicate that residue–residue connectivity is highly relevant to medicinal chemistry. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010     

A Bioorthogonal Small‐Molecule‐Switch System for Controlling Protein Function in Live Cells

Chemically induced dimerization (CID) has proven to be a powerful tool for modulating protein interactions. However, the traditional dimerizer rapamycin has limitations in certain in vivo applications because of its slow reversibility and its affinity for endogenous proteins. Described herein is a bioorthogonal system for rapidly reversible CID. A novel dimerizer with synthetic ligand of FKBP′ (SLF′) linked to trimethoprim (TMP). The SLF′ moiety binds to the F36V mutant of FK506‐binding protein (FKBP) and the TMP moiety binds to E. coli dihydrofolate reductase (eDHFR). SLF′‐TMP‐induced heterodimerization of FKBP(F36V) and eDHFR with a dissociation constant of 0.12 μM . Addition of TMP alone was sufficient to rapidly disrupt this heterodimerization. Two examples are presented to demonstrate that this system is an invaluable tool, which can be widely used to rapidly and reversibly control protein function in vivo.     

FKBP12蛋白与其抑制剂结合的分子动力学模拟和结合自由能计算

FKBP12 (FK506-binding protein-12)是一种具有神经保护和促神经再生作用的蛋白. 采用分子动力学模拟取样, 运用MM-GBSA方法计算了FKBP12和3个抑制剂(GPI-1046, 308和107)的绝对结合自由能, GPI-1046的结合能最小, 308小于107的结合能. 通过能量分解的方法考察了FKBP12蛋白的主要残基与抑制剂之间的相互作用和识别, 计算结果表明: 3个抑制剂具有相似的结合模式, Ile56和Tyr82主要表现为氢键作用, Tyr26, Phe46, Val55, Ile56, Trp59, Tyr82, Tyr87和Phe99形成疏水作用区. 计算结果和实验结果吻合.     

Selective Inhibitors of FKBP51 Employ Conformational Selection of Dynamic Invisible States

The recently discovered SAFit class of inhibitors against the Hsp90 co‐chaperone FKBP51 show greater than 10 000‐fold selectivity over its closely related paralogue FKBP52. However, the mechanism underlying this selectivity remained unknown. By combining NMR spectroscopy, biophysical and computational methods with mutational analysis, we show that the SAFit molecules bind to a transient pocket in FKBP51. This represents a weakly populated conformation resembling the inhibitor‐bound state of FKBP51, suggesting conformational selection rather than induced fit as the major binding mechanism. The inhibitor‐bound conformation of FKBP51 is stabilized by an allosteric network of residues located away from the inhibitor‐binding site. These residues stabilize the Phe67 side chain in a dynamic outward conformation and are distinct in FKBP52, thus rationalizing the basis for the selectivity of SAFit inhibitors. Our results represent a paradigm for the selective inhibition of transient binding pockets.     

Structural aspects of metal–amide complexes

An exhaustive survey of crystal structure data on simple amides and metal complexes containing monodentate amide ligands has been performed. Statistical analysis of structural features are reported as a function of the degree of alkylation of the amide functional group, the type of metal ion in the amide complex, and the type of binding to the metal ion. Average values are reported for bond lengths, bond angles, and torsional angles. Orientational preferences of the coordinated amide ligand are discussed in terms of M–O–C bond angles and M–O–C–N torsion angles.     

Synthesis of calcineurin-resistant derivatives of FK506 and selection of compensatory receptors

We used olefin metathesis to synthesize C40 derivatives of FK506 and measured their ability, when complexed to FKBP12, to inhibit calcineurin's phosphatase activity. We identified modular dimerization domains (CABs) containing segments of the calcineurin A and B polypeptides. These CABs respond to FK506 both when overexpressed in mammalian cells and in yeast or mammalian three-hybrid assays. Using chemical genetic selection, we identified compensatory mutant CABs that respond to a calcineurin-resistant FK506 derivative at concentrations well below the response threshold for CABs containing only wild-type calcineurin sequence. These reagents provide a small molecule-protein combination orthogonal to existing dimerizer systems and may be used with existing systems to increase the complexity of induced-proximity experiments. This new use of the "bump-hole" strategy protects target cells from complications arising from the inhibition of endogenous calcineurin.