There has recently been considerable interest in using NMR spectroscopy to identify ligand binding sites of macromolecules. In particular, a modular approach has been put forward by Fesik et al. (Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W. Science 1996, 274, 1531-1534) in which small ligands that bind to a particular target are identified in a first round of screening and subsequently linked together to form ligands of higher affinity. Similar strategies have also been proposed for in silico drug design, where the binding sites of small chemical groups are identified, and complete ligands are subsequently assembled from different groups that have favorable interactions with the macromolecular target. In this paper, we compare experimental and computational results on a selected target (FKBP12). The binding sites of three small ligands ((2S)1-acetylprolinemethylester, 1-formylpiperidine, 1-piperidinecarboxamide) in FKBP12 were identified independently by NMR and by computational methods. The subsequent comparison of the experimental and computational data showed that the computational method identified and ranked favorably ligand positions that satisfy the experimental NOE constraints.

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...

Structural features of a 19-nucleotide RNA hairpin loop (5'-GGCGUACGUUUCGUACGCC-3'), a loop motif which occurs in eukaryotic 18S rRNA, have been derived using multidimensional heteronuclear NMR spectroscopy in combination with local conformational analysis and torsion angle distance geometry followed by restrained energy minimization. A method to obtain both the 3JC4'P3' and 3JC4'P5' coupling constants from a set of spin-echo difference constant time HSQC spectra is introduced, and it is shown how these couplings can be assigned to the backbone angles beta and epsilon. A total of 280 distance constraints as well as 132 homo- and heteronuclear three-bond scalar coupling constants were derived from the NMR data. The structure which has been determined is a pentaloop rather than a triloop with no base pairing between G8 and C12. G8 is pointed to the minor groove where it forms a base triplet with C7-G13 that is further stabilized by hydrogen bonding to the 2'-hydroxyl group of C7. C12 is directed to the major groove where its conformation is stabilized by hydrogen bonding between O2 and HO2'. The NMR data suggest two possible, interconverting conformations with stacking of bases U10-G8 or U11-C7. Overall, the loop provides a variety of interaction sites for RNA or protein interactions.

An approach for the simultaneous acquisition of HCN and HCP as well as HCN-CCH-TOCSY and HCP-CCH-TOCSY triple resonance data sets for 13C-/15N-labelled RNAs is presented. The new HCN-CCH-TOCSY scheme unambiguously links all sugar resonances to the base nitrogen. In addition, simultaneous acquisition of HCN-CCH-TOCSY and HCP-CCH-TOCSY data sets provides sequential and base-type information in a single experiment, thereby saving data acquisition time as well as providing complementary data sets that are useful in clarifying ambiguous assignments. Virtually complete sequence-specific phosphate-ribose 1H, 31P, and base 15N1,9, assignments as well as partial 13C assignments could be obtained in a single experiment for a 0.5-mM sample of a 19-mer ribonucleotide.

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Ribonucleic acids (RNA) and RNA−protein complexes are essential components of biological information transfer, catalytic processes and are associated with regulatory functions. This broad range of biological functions is paralleled at the conformational level by a large number of non-canonical structural elements or sequences with non-standard backbone conformations, e.g., loops, bulges, pseudo-knots and complex tertiary folds. NMR spectroscopy has evolved to a powerful tool for the determination of ribonucleic acid structures of up to 20 kDa. Uniform or selective stable isotope labelling aids in solving assignment problems arising from the inherently limited chemical shift dispersion and overlap of resonances for larger nucleotide sequences. Recent developments of multi-dimensional heteronuclear NMR pulse sequences allow e.g., to directly observe the hydrogen bonding pattern of canonical Watson−Crick base pairs as well as of unusual types of base pairs, thereby opening up a fast access to secondary structure screening of RNA. Detailed conformational descriptions are obtained using conventional NOE andJcoupling-derived data, nowadays supplemented by information from residual dipolar couplings. The latter method also provides a new means for the probing of dynamical features of ribonucleic acids.

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