Crystal structure of the catalytic and ubiquitin-associated domains of the protein kinase MARK2 / PAR-1 from Rattus norvegicus (Berkenhout, 1769)

MAP/microtubule affinity regulating kinases (MARKs) are a family of protein serine/threonine kinases which have been identified by their ability to phosphorylate the microtubule-associated proteins tau, MAP2 and MAP4. Phosphorylation of the neuronal MAP tau on S262 dramatically reduces its microtubule binding capacity and leads to the formation of neurofibrillary tangles, which is a hallmark of Alzheimer’s disease. Homologues of MARK include the kinase PAR-1 in C. elegans and D. melanogaster and KIN-1 in S. cerevisiae which are involved in the generation of embryonic polarity and cell morphology respectively. Compared to other kinases, MARK is a relatively large protein (~720 amino acids) which contains an N-terminal leader sequence, a typical kinase catalytic domain, an ubiquitin associated domain (UBA), a spacer and a tail domain containing the KA1 (kinase associated) motif characteristic for this family of kinases ending with the ELKL motif. A stable fragment of MARK2 was identified by limited proteolysis, which contains the catalytic and ubiquitin associated domains. This fragment was crystallized in several variants. Crystals of the wild type construct and of two inactive mutants K82R and T208A/S212A were analyzed. K82 is essential for catalysis, T208 is the primary phosphorylation site in the activation loop which controls access of the substrate and S212 was also found to be phosphorylated in MARK2 from brain. The three variants and the selenomethionine labelled protein were crystallized in the hexagonal space group P61. Two distinct crystal forms were observed which differ by the length of the c-axis: 106.0 ± 0.37 Å (sd, n = 15) for the double mutant, 99.7 ± 0.23 Å (sd, n = 8) for the wild type and the K82R mutant; the wild type was found in both forms. All crystal structures are similar in crystal packing and folding of the molecules. The structure of the catalytic domain shows the small and large lobes typical of kinases. The substrate cleft between the lobes is wide open both in the inactivated and the wild type structures. In the crystal, two kinase moieties form a dimer, facing each other with the catalytic cleft such that helix G of one molecule inserts into the cleft of the other, similar to the dimers of phosphorylase kinase. This prevents cleft closure and a conformation reminiscent of the active state. The UBA domain is attached via a taut linker to the large lobe of the kinase domain and leans against a hydrophobic patch on the back of the small lobe. The UBA structure is unusual in that the orientation of its third helix is inverted, relative to previous structures. The ubiquitin-binding interface is partially masked by the interaction with the kinase domain, implying that detachment and/or conformational changes are necessary for activation of ubiquitin-dependent signaling. The linker sequence also contains a peptide motif similar to the "common docking domain" of the MAP kinase family, which could serve as an anchor for partners in the signaling cascade of MARK/PAR-1.