Solution structure of FK506 bound to FKBP‐12

Lepre, Christopher A.; Thomson, John A.; Moore, Jonathan M.

doi:10.1016/0014-5793(92)80292-o

Cited by 38 publications

(41 citation statements)

References 37 publications

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“…However there is only insignificant structural difference (rmsd = 0.049 nm) between free rapamycin and that bound to hFKl3P-12 (van Duyne et al, 1993). In solution, FK506 bound to FKBP-12 has a similar structure to that found in the crystal state except some modifications of the ally1 part which could be due to crystal vs. solvent effects (Lepre et al, 1992).…”

Section: The Structure Of Fkbp-12 and Its Complex With Fk506 Or Rapammentioning

confidence: 79%

Peptidylproline cis‐trans‐isomerases: immunophilins

Gałat

1993

European Journal of Biochemistry

336

184

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Two sequence-unrelated families of proteins possess peptidylproline cis-trans-isomerase activities (PPIase). PPIases are highly sequence conserved and multifunctional proteins which are present in many types of cells with a considerably divergent phylogenetic distribution. On the cellular level, PPIases occur in every compartment, both as free species and anchored to membranes. Diverse posttranslational modifications such as glycosylation, N-terminal modifications and phosphorylation constitute the additional functional features of PPIases. Folding, assembly and trafficking of proteins in the cellular milieu are regulated by PPIases. These enzymes accelerate the rate of in-vitro protein folding and they have the ability to bind proteins and act as chaperones. Some PPIases are coregulatory subunits of molecular complexes including heat-shock proteins, glucocorticoid receptors and ion channels. Secreted forms of PPIases are inflammatory and chemotactic agents for monocytes, eosinophils and basophils. The potent and clinically useful immunosuppressants CsA, FK.506 or rapamycin bind with high affinities to PPIases (immunophilins). The binding criterion allows us to sort the PPIases for the following two superfamilies of proteins : the cyclophilins (CsA-binding proteins) and the FKBP (FK.506/rapamycin-binding proteins).Although none of PPIases appeared to be essential for the viability of haploid yeast cells some of the immunophilidimmunosuppressant complexes are toxic both for yeast and mammalian cells. At least seven unlinked genes of cyclophilins and four unlinked genes of FKBP exist in human genomic DNA. Selected immunophilins regulate two different signalling pathways in lymphoid cells, namely the secretion of growth factors by stimulated T-cells and interleukin-2-induced T-cell proliferation. Moreover, selected FKBP mediate the cytotoxic effects of rapamycin in non-lymphoid cells. Accounts of the discovery of PPIases (immunophilins) and their functions are given in this review. A larger spectrum of proteins is analysed in relation to various signal-transduction pathways in lymphoid cells which involve immunophilins or their complexes with the immunosuppressants CsA, FK.506 or rapamycin.

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Section: The Structure Of Fkbp-12 and Its Complex With Fk506 Or Rapammentioning

confidence: 79%

Peptidylproline cis‐trans‐isomerases: immunophilins

Gałat

1993

European Journal of Biochemistry

336

184

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“…Although the energy refinement selects the orientation shown with the methoxy out of plane, the two conformers differ by less than 1 kcal in restraint energy and one additional restraint violation of less than 0.1 Å. As we have noted before, [33][34][35] energy refinement in the absence of the receptor protein can result in conformations dictated by the force field for the free ligand, and it is possible that stabilizing interactions with the protein might favor conformations which are higher in energy in the absence of a protein model. Therefore the alternative methoxy orientation should not be ruled out.…”

Section: Figurementioning

confidence: 94%

“…In addition, isotope editing experiments with a 13 C-labeled FK506 drug molecule permitted several studies with FKBP-12 and mutant FKBP-12 and FKBP-13 proteins that helped elucidate the structural nuances of FK506 binding and provided insight into recognition of the FKBP-12/FK506 complex by its downstream target, calcineurin. [33][34][35]37 Examples from several of these latter studies illustrate the significant value and utility of nmr isotope editing and filtering studies in drug design.…”

Section: Figure 15 Figure 16mentioning

confidence: 96%

“…The best distance geometry structures (top), based on the value of the residual error, were refined by MD in Discover (MSI, San Diego, CA) using a heating/annealing protocol as described previously. 33 The MD family (bottom) represents a subset of the refined DG structures possessing low total energy, lowest restraint energies, and fewest restraint violations (no violations Ͼ 0.06 A). Note the structures represent only one of two possible mirror image distance geometry structures that arise due to the twofold symmetry of MPA and represent equally probable solutions to the structure.…”

Section: Figurementioning

confidence: 99%

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NMR techniques for characterization of ligand binding: Utility for lead generation and optimization in drug discovery

Moore

1999

Biopolymers

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Over the last ten years, nmr spectroscopy has evolved into an important discipline in drug discovery. Initially, nmr was most useful as a technique to provide structural information regarding protein drug targets and target–ligand interactions. More recently, it has been shown that nmr may be used as an alternative method for identification of small molecule ligands that bind to protein drug targets. High throughput implementation of these experiments to screen small molecule libraries may lead to identification of potent and novel lead compounds. In this review, we will use examples from our own research to illustrate how nmr experiments to characterize ligand binding may be used to both screen for novel compounds during the process of lead generation, as well as provide structural information useful for lead optimization during the latter stages of a discovery program. © 1999 John Wiley & Sons, Inc. Biopoly 51: 221–243, 1999

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“…All 13C-'H sites that undergo exchange broadening in the present study correlate well with sites that have been determined in previous studies to possess moderate to high solvent exposure in the binary FKBP-12/I3C-FK506 complex (Lepre et al, 1993) a All data were collected on a Bruker DMX-500 spectrometer at 303 K using 0.3 mM 1: 1 binary complex and 0.3 mM 1 : 1: 1 : 1 quaternary complex in 50 mM Tris/D,O, I mM MgCI,, 1 mM CaCI,, 1 mM P-mercaptoethanol at pD = 8.2. I3C-labeled FK506 was prepared as described previously (Lepre et al, 1992).…”

mentioning

confidence: 99%

Dynamic NMR studies of ligand‐receptor interactions: Design and analysis of a rapidly exchanging complex of FKBP‐12/FK506 with a 24 kDa calcineurin fragment

et al. 1996

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Dynamic NMR methods, such as differential line broadening and transferred NOE spectroscopy, are normally reserved for the study of small molecule ligand interactions with large protein receptors. Using a combination of isotope labeling and isotope edited NMR, we have extended these techniques to characterize interactions of a much larger protein/drug complex, FKBP-I2/ FK506 with its receptor protein, calcineurin. In order to examine this multicomponent system by dynamic NMR methods, the 93 kDa, tightly bound FKBP-I2/FK506/Cn complex was replaced with a lower affinity, rapidly exchanging system consisting of FKBP-I2/FK506 (13 kDa), recombinant calcineurin subunit B (CnB) (20 kDa), and a synthetic peptide (4 kDa) corresponding to the B binding domain (BBD) of calcineurin catalytic subunit A (CnA).Analysis Complexes of the potent immunosuppressants FK506 (Fig. 1) and cyclosporin A with the immunophilins FKBP-12 and cyclophilin, respectively, have been demonstrated to inhibit signal transduction pathways leading to T-cell activation by binding to and inhibiting the protein phosphatase, calcineurin (Cn) (Liu et al., 1991). Since the immunophilin/drug complex and not the drug or immunophilin protein alone is the inhibitory species, information regarding the interaction of this complex with its downstream target, calcineurin, is considered critical for the structure-based design of improved immunosuppressive agents. Unfortunately, the ternary complexes of both FKBP-12IFK506 and Cyp/CSA with calcineurin are both large (>93 kDa) and preclude the use of NMR techniques for a direct structure determination. Therefore, we have focused upon alternative methods to probe the interaction of FKBP-l2/FK506 with calcineurin. The 93 kDa, tightly bound FKBP-l2/FK506/Cn complex was replaced with a lower affinity, rapidly exchanging system consisting of FKBP-12/FK506 (13 kDa), recombinant calcineurin subunit B (CnB) (20 kDa), and a synthetic peptide (4 kDa) corresponding to the B binding domain (BBD) of calcineurin catalytic subunit A (CnA) (Fig. 2) Dynamic NMR methods, such as differential line broadening and transferred NOE spectroscopy, normally reserved for the study of small molecule interactions with large protein receptors (reviewed in Ni, 1994), have been extended to characterize interactions of a much larger proteiddrug complex, FKBP-IUFK506 with a 24 kDa fragment of its receptor protein, calcineurin. Differential line broadening effects and additional NOE contacts for FK506 in the FKBP-I2/FK506/CnB/BBD quaternary complex versus the binary FKBP-l2/FK506 complex have been observed, and indicate which regions of the FK506 molecule are most influenced by interaction with calcineurin.

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Solution structure of FK506 bound to FKBP‐12

Cited by 38 publications

References 37 publications

Peptidylproline cis‐trans‐isomerases: immunophilins

Peptidylproline cis‐trans‐isomerases: immunophilins

NMR techniques for characterization of ligand binding: Utility for lead generation and optimization in drug discovery

Dynamic NMR studies of ligand‐receptor interactions: Design and analysis of a rapidly exchanging complex of FKBP‐12/FK506 with a 24 kDa calcineurin fragment

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