Various solution studies were conducted to address the discrepancy in the quaternary structure of AK which revealed that the formation of the cooperative tetramer is possible upon effector binding  and . Despite the fact that the enzyme had been crystallized in the absence of lysine, the structure reveals lysine bound form of CaAK which enable us to identify the key elements which are responsible for the large conformational changes associated with the inhibitor binding. The DynDom analysis clearly indentified the bending residues at the domain crossover regions (D208–L213
and E237–I250) in order to support the domain motion between MAPK inhibitor the regulatory and catalytic domains of CaAK ( Fig. 4A and B). The analysis provides the rotation angle of monomers B, D, E, I as 7.3°; 8.2°; 7.3° and 3.7°, respectively whereas no rotational angle was detected for the monomers C, F, G, H, J, K and
L when monomer A was used as the reference structure. Further rotational analysis on all combinations of monomers showed the rotational angle and the value lies between 4° to 8° between the monomers. The domain reorientation is mainly controlled by interaction between the residues K232, R235, E236, S238, Y239, H246 and E247 of catalytic domain and E303, L306, N308, V335, D336 and S337 of regulatory domains. The varied interaction is induced by either lysine binding at the homodimeric interface or nucleotide binding/release at the domain crossover regions. In order to support this observation, the relative reorientation of the domains is observed in different MjAK complex structures (PDB Ids 3C1N, 3C20 and 3C1M). The rotational www.selleckchem.com/products/gsk1120212-jtp-74057.html angle varies between 6.3° and 18.9° and demonstrates the inhibitor, substrate and cofactor binding to mjAK induces the conformational changes
between the domains. Both the CaAK and MjAK structures have shortened latch loop regions (CaAK: E343–D348 and MjAK: S366–V370) and do not appear to play a role in conformational arrangements. In contrast, the crystal structures of EcAKIII solved in both R- and T-state conformation (PDB Ids 2J0X and 2J0W) demonstrated the largest rotation (∼36.3°) between the catalytic and regulatory domain. The critical latch loop (D354–T364) leading Bumetanide to the transition from R- to T-state and tetramer formation that undergoes major rotational rearrangements. The latch loop is well conserved in the structure of AtAK (D387–I397) appears to play a role in conformational rearrangements and tetermer formation similar to EcAKIII. The superposition of four ACT domains of CaAK dimer on the corresponding four ACT domains of dimeric structures of EcAKIII (PDB 2J0X and 2J0W with rmsd of 1.3 Å and 1.5 Å, respectively), AtAK (PDB 2CDQ with rmsd of 4 Å), MjAK (PDB 3 C1 M, 3 C1 N and 3C20 with rmsd of 2 Å; 1.9 Å and 1.8 Å, respectively) revealed that ACT domains adopt a similar conformation.