Marco Betz

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.

Biomolecular NMR: a chaperone to drug discovery

Biomolecular NMR now contributes routinely to every step in the development of new chemical entities ahead of clinical trials. The versatility of NMR — from detection of ligand binding over a wide range of affinities and a wide range of drug targets with its wealth of molecular information, to metabolomic profiling, both ex vivo and in vivo — has paved the way for broadly distributed applications in academia and the pharmaceutical industry. Proteomics and initial target selection both benefit from NMR: screenings by NMR identify lead compounds capable of inhibiting protein–protein interactions, still one of the most difficult development tasks in drug discovery. NMR hardware improvements have given access to the microgram domain of phytochemistry, which should lead to the discovery of novel bioactive natural compounds. Steering medicinal chemists through the lead optimisation process by providing detailed information about protein–ligand interactions has led to impressive success in the development of novel drugs. The study of biofluid composition — metabonomics — provides information about pharmacokinetics and helps toxicological safety assessment in animal model systems. In vivo, magnetic resonance spectroscopy interrogates metabolite distributions in living cells and tissues with increasing precision, which significantly impacts the development of anticancer or neurological disorder therapeutics. An overview of different steps in recent drug discovery is presented to illuminate the links with the most recent advances in NMR methodology.

Sequence-specific assignment of histidine and tryptophan ring 1H, 13C and 15N resonances in 13C/15N- and 2H/13C/15N-labelled proteins

Methods are described to correlate aromatic 1Hδ 2/13Cδ 2 or 1Hε 1/15Nε 1 with aliphatic 13Cβ chemical shifts of histidine and tryptophan residues, respectively. The pulse sequences exclusively rely on magnetization transfers via one-bond scalar couplings and employ [15N, 1H]- and/or [13C, 1H]-TROSY schemes to enhance sensitivity. In the case of histidine imidazole rings exhibiting slow HN-exchange with the solvent, connectivities of these proton resonances with β-carbons can be established as well. In addition, their correlations to ring carbons can be detected in a simple [15N, 1H]-TROSY-H(N)Car experiment, revealing the tautomeric state of the neutral ring system. The novel methods are demonstrated with the 23-kDa protein xylanase and the 35-kDa protein diisopropylfluorophosphatase, providing nearly complete sequence-specific resonance assignments of their histidine δ-CH and tryptophan ε-NH groups.

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.