杜氏盐藻类钙调素蛋白1.6(?)晶体结构研究及KCBP马达区的表达、纯化与结晶
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摘要
钙调素是一种高度保守的酸性蛋白质,普遍存在于真核生物细胞内。它可以感受ca2+浓度变化,在信号传导通路中将Ca2+浓度的改变直接或间接的传递给效应蛋白,实现对基因表达、蛋白质合成、细胞凋亡、神经递质传递、肌肉收缩、激素合成等众多生理过程的调节。随着结构生物的发展,PDB数据库中已经提交了包括原生生物、植物和动物在内的钙调素、钙调素突变体及钙调素和靶蛋白复合物的三维结构,阐述了钙调素发挥生理功能的结构基础。高等生物中通常存在多种钙调素,结构上的细微差别使它们具有不同的生理功能。藻类钙调素报道已久,但至今未见有三维结构报道。
本研究中我们构建了杜氏盐藻类钙调素蛋白的原核表达载体,并在大肠杆菌BL2 1(DE3)中表达该蛋白。依次通过疏水层析、离子交换层析和凝胶过滤层析,获得了满足生长晶体纯度的蛋白样品。20 mg/ml的目的蛋白在4mMCaCl2,48% MPD,15%乙醇,0.1 M乙酸-乙酸钠缓冲液(pH5.5),4℃生长二周后获得适合于X射线衍射的针状晶体。捞取晶体冻于液氮,利用上海同步辐射光源进行衍射。HKL2000软件分析处理收集的数据,确定出此条件下生长出的杜氏盐藻类钙调素蛋白晶体的空间群为P21212型。晶胞参数：a=72.667 A,b=79.426 A,c=28.359 A,α=β=γ=90°以1GGZ为模型,分子置换法获得相角,用CCP4、CNS软件包和COOT软件解析结构。最终获得分辨率为1.6 A的三维结构模型。
该模型中包含了24位-162位的氨基酸,165个H20,4个Ca2+和1个MPD。R因子为0.228,Rfree因子为0.259。模型的平均温度因子为28.747A2,主链平均温度因子为25.967 A2,侧链平均温度因子为31.610 A2,具有良好的立体化学性质。从整体上讲该结构与其他物种的钙调素无较大差别,均呈哑铃型。但是中央螺旋较大的柔性使两端“球形区”的取向与其他钙调素模型存在差异。N端疏水凹槽内含有3个Met,这相同于植物不同于动物；而C端疏水凹槽内含有4个Met,这又相同于动物不用于植物。因此我们根据Met的分布推断杜氏盐藻类钙调素蛋白与靶蛋白的亲和力可能介于动物和植物之间。
驱动蛋白是分子马达的一种,整个分子分为头部、躯干和尾部。头部马达区的构象会伴随着ATP的水解发生改变,将化学能转化为机械能,拖动细胞内的“货物”沿微管定向运动。驱动蛋白在囊泡运输、染色体分离、细胞器定位等生命活动中发挥重要功能。类驱动蛋白钙调素结合蛋白隶属于驱动蛋白-14家族,为负向运输型驱动蛋白。已经证实高等生物体内钙调素可以与该蛋白相互作用。土豆类驱动蛋白钙调素结合蛋白C末端区域的晶体结构已经获得解析。然而藻类该区域的晶体结构一直未见报道,更没有该区域与钙调素复合物的晶体结构报道。
我们已经获得杜氏盐藻类驱动蛋白钙调素结合蛋白的氨基酸序列,为了进一步研究藻类驱动蛋白的特点,构建了杜氏盐藻类驱动蛋白钙调素结合蛋白马达区的原核表达载体、C末端的原核表达载体和C末端突变体的原核表达载体。16℃诱导大肠杆菌BL21(DE3)表达目的蛋白。依次使用亲和层析、离子交换层析、凝胶过滤层析,获得满足生长晶体纯度的蛋白样品。三种蛋白独立使用商业化试剂盒大规模搜索结晶条件,但是未能获得蛋白质晶体。凝胶过滤层析、pull-down和ITC证实杜氏盐藻的类驱动蛋白钙调素结合蛋白可以和类钙调素蛋白相互作用。并且ITC结果显示类驱动蛋白钙调素结合蛋白与类钙调素蛋白的作用比例可能为2：1。杜氏盐藻类驱动蛋白钙调素结合蛋白的C末端与类钙调素蛋白复合物搜索了大量结晶条件,未能获得复合物的晶体。
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.
本研究中我们构建了杜氏盐藻类钙调素蛋白的原核表达载体,并在大肠杆菌BL2 1(DE3)中表达该蛋白。依次通过疏水层析、离子交换层析和凝胶过滤层析,获得了满足生长晶体纯度的蛋白样品。20 mg/ml的目的蛋白在4mMCaCl2,48% MPD,15%乙醇,0.1 M乙酸-乙酸钠缓冲液(pH5.5),4℃生长二周后获得适合于X射线衍射的针状晶体。捞取晶体冻于液氮,利用上海同步辐射光源进行衍射。HKL2000软件分析处理收集的数据,确定出此条件下生长出的杜氏盐藻类钙调素蛋白晶体的空间群为P21212型。晶胞参数：a=72.667 A,b=79.426 A,c=28.359 A,α=β=γ=90°以1GGZ为模型,分子置换法获得相角,用CCP4、CNS软件包和COOT软件解析结构。最终获得分辨率为1.6 A的三维结构模型。
该模型中包含了24位-162位的氨基酸,165个H20,4个Ca2+和1个MPD。R因子为0.228,Rfree因子为0.259。模型的平均温度因子为28.747A2,主链平均温度因子为25.967 A2,侧链平均温度因子为31.610 A2,具有良好的立体化学性质。从整体上讲该结构与其他物种的钙调素无较大差别,均呈哑铃型。但是中央螺旋较大的柔性使两端“球形区”的取向与其他钙调素模型存在差异。N端疏水凹槽内含有3个Met,这相同于植物不同于动物；而C端疏水凹槽内含有4个Met,这又相同于动物不用于植物。因此我们根据Met的分布推断杜氏盐藻类钙调素蛋白与靶蛋白的亲和力可能介于动物和植物之间。
驱动蛋白是分子马达的一种,整个分子分为头部、躯干和尾部。头部马达区的构象会伴随着ATP的水解发生改变,将化学能转化为机械能,拖动细胞内的“货物”沿微管定向运动。驱动蛋白在囊泡运输、染色体分离、细胞器定位等生命活动中发挥重要功能。类驱动蛋白钙调素结合蛋白隶属于驱动蛋白-14家族,为负向运输型驱动蛋白。已经证实高等生物体内钙调素可以与该蛋白相互作用。土豆类驱动蛋白钙调素结合蛋白C末端区域的晶体结构已经获得解析。然而藻类该区域的晶体结构一直未见报道,更没有该区域与钙调素复合物的晶体结构报道。
我们已经获得杜氏盐藻类驱动蛋白钙调素结合蛋白的氨基酸序列,为了进一步研究藻类驱动蛋白的特点,构建了杜氏盐藻类驱动蛋白钙调素结合蛋白马达区的原核表达载体、C末端的原核表达载体和C末端突变体的原核表达载体。16℃诱导大肠杆菌BL21(DE3)表达目的蛋白。依次使用亲和层析、离子交换层析、凝胶过滤层析,获得满足生长晶体纯度的蛋白样品。三种蛋白独立使用商业化试剂盒大规模搜索结晶条件,但是未能获得蛋白质晶体。凝胶过滤层析、pull-down和ITC证实杜氏盐藻的类驱动蛋白钙调素结合蛋白可以和类钙调素蛋白相互作用。并且ITC结果显示类驱动蛋白钙调素结合蛋白与类钙调素蛋白的作用比例可能为2：1。杜氏盐藻类驱动蛋白钙调素结合蛋白的C末端与类钙调素蛋白复合物搜索了大量结晶条件,未能获得复合物的晶体。
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.
引文
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