Nitrogen-15 NMR relaxation studies of the FK506 binding protein: Backbone dynamics of the uncomplexed receptor

Cheng, Jya Wei; Lepre, Christopher A.; Chambers, Stephen P.; Fulghum, John R.; Thomson, John A.; Moore, Jonathan M.

doi:10.1021/bi00086a004

Cited by 65 publications

(87 citation statements)

References 69 publications

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“…15 N relaxation parameters were measured as described previously by Barbato et al [34], except that water suppression was achieved by watergate. 15 N dimension. To ensure an almost complete recovery of the magnetization, recycle delays of 4.5, 3.8 and 4 s were employed in the T 1 , T 2 and NOE experiments, respectively.…”

Section: Nmr Spectroscopymentioning

confidence: 99%

“…Perturbations of the amide resonances of uniformly 15 N-labelled proteins upon complexation with a ligand or another protein N) $ 0.2 p.p.m., are detected for residues located in four stretches of the primary sequence: residues 24±30, 36±41, 48±65 and 99±100. In the 3D structure of FKBP12 these residues cluster around a hydrophobic cavity already established as the FK506 binding pocket (Fig.…”

Section: Chemical Shift Changes Upon Ligand Bindingmentioning

confidence: 99%

“…A total of 50 intramolecular and 18 intermolecular distance restraints was detected in 2D 15 N/ 13 C-purged NOESY spectra. Intramolecular NOEs included 16 restraints between protons in the three-bond distance, 20 between protons with a distance of four to five bonds and 14 distance restraints between protons having a distance of more than five bonds.…”

Section: Structure Determinationmentioning

confidence: 99%

“…In contrast, the authors were careful in the interpretation of the slowfrequency motions as these terms are not treated explicitly by the`model-free' approach but are derived in the course of an optimization of the overall fit for all relaxation parameters. (We note that the N15 relaxation data published by Chang et al [15,16] were later subjected to the`quasi spectral density function' analysis [52], which is equivalent to the reduced spectral density approach used in our study. )…”

Section: Dynamical Effects Of Ligand Bindingmentioning

confidence: 99%

“…N-labelled and 15 N, 13 C-labelled FKBP12 was purified from Escherichia coli cells grown on minimal media containing 15 N ammonium chloride alone or in combination with 13 C 6 d-glucose (Martek Bioscences Corp.), respectively [17]. The protein purity was .…”

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confidence: 99%

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Solution structure of a neurotrophic ligand bound to FKBP12 and its effects on protein dynamics

Sich¹,

Improta²,

Cowley³

et al. 2000

European Journal of Biochemistry

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The structure of a recently reported neurotrophic ligand, 3-(3-pyridyl)-1-propyl(2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidinecarboxylate, in complex with FKBP12 was determined using heteronuclear NMR spectroscopy. The inhibitor exhibits a binding mode analogous to that observed for the macrocycle FK506, used widely as an immunosuppressant, with the prolyl ring replacing the pipecolyl moiety and the amide bond in a trans conformation. However, fewer favourable protein±ligand interactions are detected in the structure of the complex, suggesting weaker binding compared with the immunosuppressant drug. Indeed, a micromolar dissociation constant was estimated from the NMR ligand titration profile, in contrast to the previously published nanomolar inhibition activity. Although the inhibitor possesses a remarkable structural simplicity with respect to FK506, 15 N relaxation studies show that it induces similar effects on the protein dynamics, stabilizing the conformation of solvent-exposed residues which are important for mediating the interaction of immunophilin/ ligand complexes with molecular targets and potentially for the transmission of the neurotrophic action of FKBP12 inhibitors.Keywords: FKBP12; ligand binding; nerve regeneration; NMR structure; protein dynamics.For several years there has been major interest in the development of inhibitors of the FK506 binding protein (FKBP12) because of their potential immunosuppressive effects [1±4]. Recently, FKBP12 was also identified as a target for the promotion of neurite outgrowth [5] and neurotrophic and neuroprotective effects could be associated with the action of different FKBP12 ligands [6,7]. These findings have extended the therapeutic potential of FKBP12-binding drugs to the treatment of nerve injuries and neurodegenerative disorders such as Alzheimer's and Parkinson's diseases.While it is well known that the immunosuppressive effects of FKBP12 are mediated by the interaction of the drug± immunophilin complex with calcineurin, the mechanism of the neuronal activity of FKBP12 has not yet been elucidated. However, it has been shown that the neurotrophic properties of immunophilin ligands are independent of their immunosuppressive action [8] and much effort has been directed toward the design of FKBP12-binding molecules, which are neurotrophic but nonimmunosuppressant, for clinical use [9,10]. One of the most promising compounds was discovered by scientists at Guilford Pharmaceuticals (1, Fig. 1; alternative name: GPI-1046). This ligand was synthesized within a series of small molecules that mimic the FKBP12-binding portion of FK506, a well-known immunosuppressant drug (Fig. 1), but lack the structural requirements for calcineurin inhibition. Among this series, 1 was found to be remarkably potent in inhibiting the peptidyl-prolyl cis±trans isomerase (PPIase) activity of FKBP12 and in promoting nerve regeneration both in vitro and in vivo [11].Given the therapeutic relevance of neurotrophic nonpeptidic molecules, we investigated the interaction of 1 and FKB...

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Section: Nmr Spectroscopymentioning

confidence: 99%

Section: Chemical Shift Changes Upon Ligand Bindingmentioning

confidence: 99%

Section: Structure Determinationmentioning

confidence: 99%

Section: Dynamical Effects Of Ligand Bindingmentioning

confidence: 99%

mentioning

confidence: 99%

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Solution structure of a neurotrophic ligand bound to FKBP12 and its effects on protein dynamics

Sich¹,

Improta²,

Cowley³

et al. 2000

European Journal of Biochemistry

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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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NMR studies of the structure and dynamics of peptide E, an endogenous opioid peptide that binds with high affinity to multiple opioid receptor subtypes

1999

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Nitrogen-15 NMR relaxation studies of the FK506 binding protein: Backbone dynamics of the uncomplexed receptor

Cited by 65 publications

References 69 publications

Solution structure of a neurotrophic ligand bound to FKBP12 and its effects on protein dynamics

Solution structure of a neurotrophic ligand bound to FKBP12 and its effects on protein dynamics

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

NMR studies of the structure and dynamics of peptide E, an endogenous opioid peptide that binds with high affinity to multiple opioid receptor subtypes

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