The 1.6(?) Crystal Structure Of Calmodulin-like Protein And Expression, Purification And Crystallization Of KCBP Motor Domain From Dunaliella Salina

Calmodulin (CaM) is a highly conserved acidic protein, and ubiquitous in all eukaryotic cells. As a Ca2+ sensor in signaling pathways, it can directly or indirectly affect the responsive proteins when the concentration of Ca2+ in cytosol is changed. So it plays an important role in gene expression, protein synthesis, cell apoptosis, neurotransmitter transmission, muscle contraction, hormone synthesis and other physiological processes. With the development of structure biology, a large number of three-dimensional structures of CaM and mutants and the complex of CaM with target protein from protists, plants and animals have been demonstrated and submitted to PDB. The structural basis of their physiological functions has been described. There are several kinds of CaM with minute difference that implement them the capacity in different physiological functions in higher plants. Although CaM from algae has been reported earlier, its three-dimensional structure has not yet been reported.In the present study, therefore, we constructed the expression vector of Dunaliella salina calmodulin-like protein (DSCLP) and expressed the protein in E. coli BL21 (DE3). Subsequently, by using hydrophobic chromatography, ion exchange chromatography and gel filtration chromatography successively, the proteins which could be used for crystallization were obtained. The needle crystals suitable for X-ray diffraction were harvested after they were incubated in DSCLP of 20 mg/ml 4 mM CaCl2,48% MPD,15% ethanol,0.1 M acetic acid-sodium acetate buffer (pH5.5), at 4℃for two weeks. The crystals were frozen in liquid nitrogen and then diffracted on Shanghai synchrotron radiation facility. Data were collected and analyzed using HKL2000 software. The space group was P21212 with unit cell dimensions:a=72.667 A, b=79.426A, c=28.359 A, andα=β=γ=90°. The phase was determined by molecular replacement with 1GGZ. The refinement of the DSCLP structure was performed using CCP4, CNS and COOT. Finally we obtained the three-dimensional model at 1.6 A resolution. The model contains 24-162 amino acids. The final crystallographic R factor was 0.228 and R free factor was 0.259. The average temperature factors of the model with well stereochemistry, the main chain and the side chain were 28.747 A2,25.967 A2 and 31.610 A2, respectively. The whole structure of DSCLP was the same as other species, being a dumbbell shape. The difference was that it had a great flexible central helix generating different N-lobe and C-lobe with 1GGZ. N terminal hydrophobic groove contained 3 Met which was the same as calmodulin of plants but different from that of animals, while C terminal hydrophobic groove contained 4 Met which was the same as calmodulin of animals but different from that of plants. It is suggested that the affinity of DSCLP with target proteins was between the calmodulin of animals and plants according to the distribution of Met.Kinesin is one of molecular motors with the parts of head, stalk and tail. In the motor domain of head, ATP is hydrolyzed leading to change of its conformation, consequently chemical energy is transformed into mechanical energy, dragging the "cargo" to move along microtubules. Kinesin plays a significant role in vesicle transport, chromosome segregation and organelle positioning in cell life. Kinesin-like calmodulin-binding protein (KCBP) belonged to kinesin-14 family which slides to the minus end of microtube. It has been confirmed that calmodulins of higher plants interact with and regulate KCBP in vivo. The crystal structure of KCBP C-terminal domain from potato has been solved. However, the crystal structures of this domain from algae and the complex of calmodulin and KCBP have not been reported so far.In order to further study the characteristics of algal KCBP, the prokaryotic expression vectors of the motor domain, wild C terminal domain and mutant C terminal domain of KCBP from Dunaliella sallina were constructed based on its sequence obtained at our laboratory. These proteins were expressed in E. coli BL21 (DE3) at 16℃. Followed by affinity chromatography, ion exchange chromatography and gel filtration chromatography, the purified proteins were obtained meeting the growing crystal condition. Commercial kits were used for large-scale search of crystallization conditions, but failed to obtain protein crystal. We confirmed C terminal domain of KCBP from Dunaliella sallina can interact with DSCLP by gel filtration chromatography, pull-down and ITC. And the result of ITC suggested that the interaction ratio of KCBP and DSCLP maybe 2:1. Although a large number of crystallization conditions were searched for the complex of KCBP C terminal domain and DSCLP to be crystallized, no crystal was obtained.