X‐ray Structural Analysis of Tau‐Tubulin Kinase 1 and Its Interactions with Small Molecular Inhibitors

Abstract

Tau-tubulin kinase 1 (TTBK1) is a serine/threonine/tyrosine kinase that putatively phosphorylates residues including S422 in tau protein. Hyperphosphorylation of tau protein is the primary cause of tau pathology and neuronal death associated with Alzheimer's disease. A library of 12 truncation variants comprising the TTBK1 kinase domain was screened for expression in Escherichia coli and insect cells. One variant (residues 14-313) could be purified, but mass spectrometric analysis revealed extensive phosphorylation of the protein. Co-expression with lambda phosphatase in E. coli resulted in production of homogeneous, nonphosphorylated TTBK1. Binding of ATP and several compounds to TTBK1 was characterized by surface plasmon resonance. Crystal structures of TTBK1 in the unliganded form and in complex with ATP, and two high-affinity ATP-competitive inhibitors, 3-[(6,7-dimethoxyquinazolin-4-yl)amino]phenol (1) and methyl 2-bromo-5-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)benzoate (2), were elucidated. The structure revealed two clear basic patches near the ATP pocket providing an explanation of TTBK1 for phosphorylation-primed substrates. Interestingly, compound 2 displayed slow binding kinetics to TTBK1, the structure of TTBK1 in complex with this compound revealed a reorganization of the L199-D200 peptide backbone conformation together with altered hydrogen bonding with compound 2. These conformational changes necessary for the binding of compound 2 are likely the basis of the slow kinetics. This first TTBK1 structure can assist the discovery of novel inhibitors for the treatment of Alzheimer's disease.

Identification of a Dual Inhibitor of SRPK1 and CK2 That Attenuates Pathological Angiogenesis of Macular Degeneration in Mice

“…Peptide flipping induced by inhibitor binding to the pocket, which rearranges the hydrogen-bond network, has been demonstrated to enhance the inhibitor selectivity in mitogenactivated phosphatase and t-tubulin kinase 1 (Fitzgerald et al, 2003;Nolen et al, 2003;Xue et al, 2013). SRPIN340 also induced peptide flipping in the pocket, a process in which the trifluoromethyl group of SRPIN340 may be involved.…”

Tau-tubulin kinase

“…For the crystallography of TTBK1, TTBK1 (1–313) and (14–313) fragments were expressed in E. coli and insect cells, which were found as multi-phosphorylated forms (Xue et al, 2013). To avoid this problem, TTBK1 protein was co-expressed with lambda phosphatase, by which TTBK1 was dephosphorylated and was subsequently co-crystallized with two high-affinity ATP-competitive inhibitors: 3-[(6,7-dimethoxyquinazolin-4-yl)amino]phenol (compound 1) and methyl 2-bromo-5-(7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)benzoate (compound 2).…”

The structure of human tau-tubulin kinase 1 both in the apo form and in complex with an inhibitor

“…In the recent TTBK1 structure (PDB entry 4btk; Xue et al, 2013) the gatekeeper methionine (107 in our structure; 131 in 4btk) is essentially unperturbed relative to the apo structure, and the phenol moiety of the 4btk ligand (DTQ) is perpendicular to the bromophenol moiety of compound 3. Potential reasons for this difference include (i) the hydrogen bond between the phenolic hydroxyl moiety and Glu77 and Asp176 favours this orientation, (ii) the same phenolic hydroxyl moiety is likely to clash with the stem of Lys63 if rotated into a similar orientation as the ligand in the 4btk structure, (iii) the bicyclic cores of the two ligands are not completely aligned, resulting in a slightly different vector connecting the core with the aniline N atoms of the respective substituents, and (iv) if the aniline ring of compound 3 is rotated to match the ligand structure in 4btk, the bromo moiety would potentially clash with HOH587 and HOH589, and may disrupt the hydrogen-bond network formed between these waters, HOH525 and the protein.…”

“…The hydrogen bonds that it forms to Asn159 and Asp176 are similar in geometry to those that coordinate the ion in the ATP-bound structures. Note, however, that the recent TTBK1 structure with ADP bound (PDB entry 4btj; Xue et al, 2013) does not show a Mg 2+ ion, but it would be expected to bind in a similar location coordinated by Asp176 and Asn159. As in the apo structure, the DFG motif adopts the DFG-in activated conformation and compound 3 may be classified as a type I inhibitor (Zuccotto et al, 2010).…”

“…Just prior to the submission of this paper, another report of TTBK1 structures appeared (Xue et al, 2013). A report of the crystallization of TTBK2 (Kitano-Takahashi et al, 2007) also mentions that data had been collected from a TTBK2 crystal to 2.8 Å resolution, but as of December 2013 no paper had described the TTBK2 structure nor had the coordinates been deposited in the PDB.…”