Nmr Backbone Assignment Of A Kinase

The partial backbone assignment of the catalytic domain of protein kinase A (PKA) using a combination of NMR techniques was reported in 2004 [51]. This represented the first reported NMR assignment of any protein kinase catalytic domain. Backbone resonance assignment of the 42kDa protein was achieved using a combination of triple-labelled (2H, 13C, 15N) protein with classical NMR assignment, back-calculation of chemical shifts from X-ray structures, the use of paramagnetic adenosine derivatives as spin-labels and selective amino acid labelling.

PKA is not currently a drug target due to its broad spectrum activity and multiple physiological functions. However, it is considered to be a useful protein for studying other protein kinases [53]. Several X-ray structures of PKA, with and without bound inhibitors, have been published. The structure of the non-phosphorylated, inactive structure was, however, unknown. The authors were keen to emphasise the dynamic nature of proteins, particularly the conformational changes occurring upon activation, and upon binding of substrates and inhibitors, and point out that NMR is an ideal tool for studying motional processes by relaxation analysis.

The authors expressed PKA consisting of 353 amino acids, of which eight are prolines. Resonances of 274 backbone amide peaks were visible in the spectrum, of which 191 were assigned. It was possible to assign resonances for the N- and C-terminal sequences, the majority of the N-lobe, including the glycine-rich loop, and most of the solvent-exposed residues of the C-lobe. This enabled a determination of the structure for the more flexible parts of the structure. However, many correlations were missing for the more rigid parts of the protein, resulting in incomplete data sets. It was only possible to correlate relatively short stretches of the protein, making it difficult to match on the sequence. To circumvent this difficulty, the authors adopted a novel approach of predicting chemical shifts for the backbone and Cb from known structures [54]. The experimental data from a correlated stretch of amino acids from a known structure was found to provide sufficient information to allow matching to the novel structure.

It was possible to unambiguously assign the amino acids near to the ATP-binding site by the use of paramagnetic spin-labelled adenosine (Figure 1.11), leading to a pure distance-dependent line broadening or disappearance for those signals within approximately 20 A of the spin-labelled adenosine.

Selective labelling of PKA with 15N-labelled Phe, Tyr, Leu, Asp, Ile and Val was carried out allowing more facile assignment of the peaks due to those residues. For example, it was possible to assign 19 out of the 20 resonances due to valine in the protein using this approach. 13C-labelled Tyr was used in conjunction with 15N-labelled Val allowing the identification of the only Tyr-Val sequence in PKA via a 2D HNCO spectrum. The authors were able to identify the Val-123 transverse relaxation optimised spectroscopy (TROSY) peak, which had no residual correlations in the 3D spectrum. By adding the known kinase inhibitor H7, it was possible to define the binding site by examination of the chemical shift perturbations and to map the interaction surfaces. This experiment confirmed the cry-stallographically determined position of the ligand [55].

An examination of mutant PKA proteins was undertaken. Phosphor-ylation of Thr-197 is required to activate PKA and phosphorylation of Ser-338 enhances stability of the protein. Replacement of Thr-197 and/or Ser-338 by Ala was examined to determine any conformational changes in the protein. Both single substitution mutants were expressed in Escherichia coli in similar levels to wild-type protein. However, both mutants were found to be less stable, as had been previously described. The double mutant

Fig. 1.11 Structure of spin-labelled adenosine (1-oxyl-2,2, 5, 5-tetramethylpyrroline-3-carboxylate (5-aminoadenosine) -amide).

Thr-197-Ala/Ser-338-Ala could also be expressed but was highly unstable and aggregated readily. To investigate folding of the Thr-197-Ala and Ser-338-Ala mutants, the proteins were 15N-labelled and their TRO-SY-NMR spectra were recorded. The peak pattern for the Ser-338-Ala mutant closely matched that of the wild-type protein. However, the Thr-197-Ala mutant showed significant differences in the peak patterns, thus indicating conformational changes in at least one region of the protein.

The ability to assign a significant percentage of the NMR signals from a kinase catalytic domain is an important prerequisite for the study of the conformational changes that the kinase catalytic domain undergoes upon activation, phosphorylation events, ATP binding and inhibitor binding. These experiments also open up the possibility of using techniques such as SAR by NMR with kinases in the future, thus potentially allowing the screening by NMR of compound libraries, and the discovery of novel and selective inhibitors of kinases.

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