CpCDPK2 structure CpCDPK2 comprises an N terminal domain that is pre dicted to bind a carbohydrate, KD, and the CAD. We have solved the crystal structures of CpCDPK2 KD with indirubin E804 3,4 dihydroxybutyloxy} amino 1H,2H 2,3 biindol 2 one bound, as well as the apo form, Both structures have completely ordered activation loops, with a helix C of the indirubin bound structure not completely in the activated form, There is a dramatic difference in glycine rich loops between the apo and the indirubin bound structures. In the apo form, the loop is moved up and away from the activation site, adopting a confor mation less amenable to binding ATP. With the indiru bin bound structure, the backbone atoms from the loop move up to 8 closer to the active site.
Once again, the indole moiety is interacting with the backbone hinge residues Glu103 and Cys105, The hydroxyl groups from the tail of the indiru bin form a series of H bonds with residues from the C lobe of the catalytic domain. Glu109 is pulled up by this interaction, such that the a helix D is more ordered and contains an additional turn compared to the a helix D from the i thought about this apo structure. CpCDPK4 structure The KD structure of CpCDPK4 has been solved in the presence of a non hydrolyzable ATP analogue, Sequence alignment shows that CpCDPK4 has a unique insert within the kinase domain that is particu larly cysteine rich in the centre. 101LNVFIDDSTGK CAMDVVKTQICPCPECNEEAINGSIHGFRES140, This insert is situated between the hinge region and a helix F.
It consists of an anti parallel b mesh that interacts with the helical C lobe of the KD and a helix that interacts with the N terminal b lobe, Embedded in the helix is a zinc ion coordinated by His93, Cys122, Cys124 and Cys127, This zinc finger is not present in any other known protein selelck kinase inhibitor KD, based on searches by sequence and structure, making the insert par ticularly interesting and unique. When CpCDPK4 is overlaid with the structure of PKC ? that includes the C terminal resi dues, we see that the zinc finger lies within the same vicinity as the C terminal tail of PKC ?, This tail is believed to bring a hydrophobic residue, Phe543 into van der Waals contact with the adenosine head of ATP and the tail forms H bonds with the glycine rich loop, thereby ensuring an activated state for PKC ?, The zinc finger of CpCDPK4 is in the vicinity of the N lobe and the exact volume occupied by the PKC ? C terminal tail.
Instead of a phenylalanine, however, an isoleucine is in position to maintain the more hydrophobic area for the adenosine head group. In addi tion, for human CLK1 and CLK3, the C terminal lobe bears an insertion between the two sheets b7 and b8, termed the CLK specific bhp bhp hairpin, and renders the sub strate docking groove inaccessible, Although the CLK specific hairpin partially overlaps with the b mesh portion of the CpCDPK4 insert, it is distinct in orientation and lacks sequence identity to the CpCDPK4 insert, Thirdly, although using a deletion mutant to obtain the crystal structure of human SRPK1 that truncates the N termi nus by 41 residues and eliminates a spacer of 217 resi dues, there is some overlap between the location of the remaining spacer and the CpCDPK4 insert, There are two small motifs that share sequence identity between the CpCDPK4 insert and SPRK, specifically 119TxIxxxP125 and 128NExxI132, but only the latter shares spa tial and structural similarity, forming the first a helix in each insert structure but in a nearly orthogonal orienta tion.