Bettina Elshorst

NMR solution structure of a complex of calmodulin with a binding peptide of the Ca2+ pump.

The three-dimensional structure of the complex between calmodulin (CaM) and a peptide corresponding to the N-terminal portion of the CaM-binding domain of the plasma membrane calcium pump, the peptide C20W, has been solved by heteronuclear three-dimensional nuclear magnetic resonance (NMR) spectroscopy. The structure calculation is based on a total of 1808 intramolecular NOEs and 49 intermolecular NOEs between the peptide C20W and calmodulin from heteronuclear-filtered NOESY spectra and a half-filtered experiment, respectively. Chemical shift differences between free Ca(2+)-saturated CaM and its complex with C20W as well as the structure calculation reveal that C20W binds solely to the C-terminal half of CaM. In addition, comparison of the methyl resonances of the nine assigned methionine residues of free Ca(2+)-saturated CaM with those of the CaM/C20W complex revealed a significant difference between the N-terminal and the C-terminal domain; i.e., resonances in the N-terminal domain of the complex were much more similar to those reported for free CaM in contrast to those in the C-terminal half which were significantly different not only from the resonances of free CaM but also from those reported for the CaM/M13 complex. As a consequence, the global structure of the CaM/C20W complex is unusual, i.e., different from other peptide calmodulin complexes, since we find no indication for a collapsed structure. The fine modulation in the peptide protein interface shows a number of differences to the CaM/M13 complex studied by Ikura et al. [Ikura, M., Clore, G. M., Gronenborn, A. M., Zhu, G., Klee, C. B., and Bax, A. (1992) Science 256, 632-638]. The unusual binding mode to only the C-terminal half of CaM is in agreement with the biochemical observation that the calcium pump can be activated by the C-terminal half of CaM alone [Guerini, D., Krebs, J., and Carafoli, E. (1984) J. Biol. Chem. 259, 15172-15177].

NMR backbone assignment of a protein kinase catalytic domain by a combination of several approaches: application to the catalytic subunit of cAMP-dependent protein kinase.

Protein phosphorylation is one of the most important mechanisms used for intracellular regulation in eukaryotic cells. Currently, one of the best‐characterized protein kinases is the catalytic subunit of cAMP‐dependent protein kinase or protein kinase A (PKA). PKA has the typical bilobular structure of kinases, with the active site consisting of a cleft between the two structural lobes. For full kinase activity, the catalytic subunit has to be phosphorylated. The catalytic subunit of PKA has two main phosphorylation sites: Thr197 and Ser338. Binding of ATP or inhibitors to the ATP site induces large structural changes. Here we describe the partial backbone assignment of the PKA catalytic domain by NMR spectroscopy, which represents the first NMR assignment of any protein kinase catalytic domain. Backbone resonance assignment for the 42 kDa protein was accomplished by an approach employing 1) triply (2H,13C,15N) labeled protein and classical NMR assignment experiments, 2) back‐calculation of chemical shifts from known X‐ray structures, 3) use of paramagnetic adenosine derivatives as spin‐labels, and 4) selective amino acid labeling. Interpretation of chemical‐shift perturbations allowed mapping of the interaction surface with the protein kinase inhibitor H7. Furthermore, structural conformational changes were observed by comparison of backbone amide shifts obtained by 2D 1H,15N TROSY of an inactive Thr197Ala mutant with the wild‐type enzyme.

How Much NMR Data Is Required To Determine a Protein–Ligand Complex Structure?

Here we present an NMR‐based approach to solving protein–ligand structures. The procedure is guided by biophysical, biochemical, or knowledge‐based data. The structures are mainly derived from ligand‐induced chemical‐shift perturbations (CSP) induced in the resonances of the protein and ligand‐detected saturated transfer difference signals between ligands and selectively labeled proteins (SOS‐NMR). Accuracy, as judged by comparison with X‐ray results, depends on the nature and completeness of the experimental data. An experimental protocol is proposed that starts with calculations that make use of readily available chemical‐shift perturbations as experimental constraints. If necessary, more sophisticated experimental results have to be added to improve the accuracy of the protein–ligand complex structure. The criteria for evaluation and selection of meaningful complex structures are discussed. These are exemplified for three complexes, and we show that the approach bridges the gap between theoretical docking approaches and complex NMR schemes for determining protein–ligand complexes; especially for relatively weak binders that do not lead to intermolecular NOEs.

Influence of Heparin Mimetics on Assembly of the FGF-FGFR4 Signaling Complex

Fibroblast growth factor (FGF) signaling regulates mammalian development and metabolism, and its dysregulation is implicated in many inherited and acquired diseases, including cancer. Heparan sulfate glycosaminoglycans (HSGAGs) are essential for FGF signaling as they promote FGF·FGF receptor (FGFR) binding and dimerization. Using novel organic synthesis protocols to prepare homogeneously sulfated heparin mimetics (HM), including hexasaccharide (HM6), octasaccharide (HM8), and decasaccharide (HM10), we tested the ability of these HM to support FGF1 and FGF2 signaling through FGFR4. Biological assays show that both HM8 and HM10 are significantly more potent than HM6 in promoting FGF2-mediated FGFR4 signaling. In contrast, all three HM have comparable activity in promoting FGF1·FGFR4 signaling. To understand the molecular basis for these differential activities in FGF1/2·FGFR4 signaling, we used NMR spectroscopy, isothermal titration calorimetry, and size-exclusion chromatography to characterize binding interactions of FGF1/2 with the isolated Ig-domain 2 (D2) of FGFR4 in the presence of HM, and binary interactions of FGFs and D2 with HM. Our data confirm the existence of both a secondary FGF1·FGFR4 interaction site and a direct FGFR4·FGFR4 interaction site thus supporting the formation of the symmetric mode of FGF·FGFR dimerization in solution. Moreover, our results show that the observed higher activity of HM8 relative to HM6 in stimulating FGF2·FGFR4 signaling correlates with the higher affinity of HM8 to bind and dimerize FGF2. Notably FGF2·HM8 exhibits pronounced positive binding cooperativity. Based on our findings we propose a refined symmetric FGF·FGFR dimerization model, which incorporates the differential ability of HM to dimerize FGFs.

A Combination of Spin Diffusion Methods for the Determination of Protein-Ligand Complex Structural Ensembles.

Structure-based drug design (SBDD) is a powerful and widely used approach to optimize affinity of drug candidates. With the recently introduced INPHARMA method, the binding mode of small molecules to their protein target can be characterized even if no spectroscopic information about the protein is known. Here, we show that the combination of the spin-diffusion-based NMR methods INPHARMA, trNOE, and STD results in an accurate scoring function for docking modes and therefore determination of protein-ligand complex structures. Applications are shown on the model system protein kinase A and the drug targets glycogen phosphorylase and soluble epoxide hydrolase (sEH). Multiplexing of several ligands improves the reliability of the scoring function further. The new score allows in the case of sEH detecting two binding modes of the ligand in its binding site, which was corroborated by X-ray analysis.

Folding and activity of cAMP-dependent protein kinase mutants

The catalytic subunit of cAMP‐dependent protein kinase (PKA) can easily be expressed in Escherichia coli and is catalytically active. Four phosphorylation sites are known in PKA (S10, S139, T197 and S338), and the isolated recombinant protein is a mixture of different phosphorylated forms. Obtaining uniformly phosphorylated protein requires separation of the protein preparation leading to significant loss in protein yield. It is found that the mutant S10A/S139D/S338D has similar properties as the wild‐type protein, whereas additional replacement of T197 with either E or D reduces protein expression yield as well as folding propensity of the protein. Due to its high sequence homology to Akt/PKB, which cannot easily be expressed in E. coli, PKA has been used as a surrogate kinase for drug design. Several mutations within the ATP binding site have been described to make PKA even more similar to Akt/PKB. Two proteins with Akt/PKB‐like mutations in the ATP binding site were made (PKAB6 and PKAB8), and in addition S10, S139 and S338 phosphorylation sites have been removed. These proteins can be expressed in high yields but have reduced activity compared to the wild‐type. Proper folding of all proteins was analyzed by 2D 1H, 15N‐TROSY NMR experiments.

1H, 13C and 15N assignment of D2 domain of human fibroblast growth factor receptor 4

Fibroblast growth factor receptor (FGFR) 4 has been associated with progression of melanoma, breast, head and neck and hepatocellular carcinoma and is therefore an interesting target for therapeutic intervention (Ho et al. in J Hepatol 50:118–127, 2009). The extracellular D2 domain of the FGFR4 receptor contains a heparin binding site and the main interaction site with the fibroblast growth factor. We report the sequential backbone and side chain resonance assignment of the D2 domain of human FGFR4.

MOTOR: Model assisted software for NMR structure determination

Eukaryotic proteins with important biological function can be partially unstructured, conformational flexible, or heterogenic. Crystallization trials often fail for such proteins. In NMR spectroscopy, parts of the polypeptide chain undergoing dynamics in unfavorable time regimes cannot be observed. De novo NMR structure determination is seriously hampered when missing signals lead to an incomplete chemical shift assignment resulting in an information content of the NOE data insufficient to determine the structure ab initio. We developed a new protein structure determination strategy for such cases based on a novel NOE assignment strategy utilizing a number of model structures but no explicit reference structure as it is used for bootstrapping like algorithms. The software distinguishes in detail between consistent and mutually exclusive pairs of possible NOE assignments on the basis of different precision levels of measured chemical shifts searching for a set of maximum number of consistent NOE assignments in agreement with 3D space. Validation of the method using the structure of the low molecular‐weight‐protein tyrosine phosphatase A (MptpA) showed robust results utilizing protein structures with 30–45% sequence identity and 70% of the chemical shift assignments. About 60% of the resonance assignments are sufficient to identify those structural models with highest conformational similarity to the real structure. The software was benchmarked by de novo solution structures of fibroblast growth factor 21 (FGF21) and the extracellular fibroblast growth factor receptor domain FGFR4 D2, which both failed in crystallization trials and in classical NMR structure determination. Proteins 2013; 81:2007–2022.