空肠弯曲菌血红素氧合酶ChuZ的结构解析与功能研究

发布时间：2017-12-31 16:06

本文关键词：空肠弯曲菌血红素氧合酶ChuZ的结构解析与功能研究　出处：《第三军医大学》2011年博士论文　论文类型：学位论文

【摘要】：空肠弯曲菌(Campylobacter jejuni, C. jejuni)是一种重要的食源性感染病原菌。全球范围内,空肠弯曲菌感染是急性胃肠炎最常见的病因之一,其严重的并发症为Guillain-Barré和Miller-Fischer综合症。随着日益严重的多重耐药现象的出现,C. jejuni感染的控制和治疗受到严峻的考验,迫切需要寻找新的途径来控制这类致病菌的感染。铁对于细菌的生长是至关重要的,尽管地球上有着非常丰富的铁元素,但是对于致病菌来说铁的生物利用度却相当低。对C. jejuni来说,来自宿主红细胞裂解的血红蛋白血红素(heme)是其铁的主要来源。而血红素氧合酶ChuZ在降解血红素并获取铁的机制中起到非常重要的作用。本研究拟解析ChuZ的晶体结构,从分子水平阐明C. jejuni利用铁元素的机制,从而为以ChuZ为靶点,通过阻断C. jejuni对铁元素的摄取,开发抗C. jejuni感染的的新型药物提供理论依据。方法: 1.chuZ基因克隆及重组ChuZ蛋白的制备。以C. jejuni(NCTC11168)全基因组为模板,PCR扩增chuZ基因并构建到原核表达载体pET22b上,采用E. coli BL21工程菌表达ChuZ蛋白。依次采用亲和层析、分子筛层析和离子交换层析方法纯化得到高质量的ChuZ蛋白。 2.ChuZ结晶条件搜索。采用Hampton Research公司的72孔板(HR3-086)和晶体初筛试剂盒Index、crystal screen HR110、SaltRx和PEG/ion等,通过Mircobatch法进行初步的结晶条件筛选,在得到初步结晶条件的基础上,对结晶条件进行优化获得最终的结晶条件。 3.晶体数据收集处理和结构解析。 X-射线晶体衍射方法收集到ChuZ-hemin的衍射数据,采用分子置换法以已有结构(3GAS)为模型获得相位并解析得到ChuZ-hemin复合物的晶体结构,并对最终模型进行分析。 4.酶活相关关键残基功能分析。在结构分析的基础上,制备参与结合底物血红素的相关残基突变体,对参与结合底物血红素的相关残基进行突变,检测突变体的酶学活性并与野生型ChuZ活性相比较,验证和评价ChuZ酶活的关键残基。 5.表面独特的血红素结合位点的分析。分别用紫外分光光度法和ITC法测量ChuZ蛋白及其突变体与血红素的结合常数和结合比例。 6.突变体晶体结构分析。制备参与结合底物血红素的相关残基突变体蛋白,结合hemin后采用制备ChuZ晶体同样方法、收集衍射数据并解析突变体蛋白的晶体结构。结果: 1.成功构建了表达ChuZ蛋白的重组质粒pET22b-chuZ,并实现了其以可溶形式表达。经多种层析方法的联合应用后,纯化得到了高纯度的ChuZ蛋白,蛋白纯度达到95%以上。 2.以出现针状晶体条件优化,最终晶体生长条件为:蛋白浓度168mg/ml,microbatch板生长,池液条件:0.1M MES 23%PEG400 50mM pH6.5 iminazole 50mM ammonium tartrate ,蛋白与池液1:1混合放置3天后即出现红色棍状晶体,七天后晶体大小为0.1×0.1×0.7 mm3。 3.运用X-射线晶体衍射法收集了一套2.4?分辨率的ChuZ-hemin数据,数据处理结果:空间群C2221,晶胞参数a = 106.474, b = 106.698,c = 52.464 ?,α=β=γ=90°。运用PHASER程序以HugZ(PDB ID 3GAS)为模型,采用分子置换法解析了ChuZ的三维结构,该结构采用split-barrel折叠方式,明显不同于经典的全α螺旋的单体血红素氧合酶的结构。明确了参与底物结合的关键残基如R166、H9和H14。 4.成功构建了R166A、H245Q/R166A、H245Q/H9A/H14A、R166A/H9A/H14A、H9A/H14A突变体,通过酶活实验明确了R166A/H9A/H14A完全丧失了酶的活性。 5.紫外吸光度值测定结果表明:H9A/H14A的ChuZ突变体结合一个hemin,野生型ChuZ结合1.5个hemin。ITC结果表明H9A/H14A突变体和wild-type ChuZ的结合常数与其它细菌的HO基本一致,结合比例与紫外结果和晶体结构中一致。 6.获得了H9A/H14A突变体的晶体,并收集得到一套3?的数据,电子密度图显示,其表面血红素结合位点没有血红素的残余密度,提示该突变体丧失了结合血红素的能力。结论: ChuZ的三维结构采取独特的split-barrel折叠方式,与大多数获得结构解析的全α螺旋的单体血红素氧合酶的结构明显不同,属于一个以HugZ为代表的新的血红素氧合酶家族。ChuZ在溶液和晶体中以同源二聚体存在。在ChuZ的精细结构中发现一个新的血红素结合位点,它与普遍存在于血红素蛋白中的“经典位点”不同,位于分子表面通过水分子介导的四个组氨酸结合血红素,这样的结合方式在其他血红素氧合酶中尚未发现过。定点突变研究证实了ChuZ是新一类血红素结合蛋白家族的特殊成员,并通过对ChuZ的结构-功能关系分析以及与同源蛋白的对比研究,发现C端结构域的血红素结合“口袋”是ChuZ酶催化机制的关键结构基础。
[Abstract]:Campylobacter jejuni (Campylobacter jejuni, C. jejuni) is an important foodborne pathogens. Worldwide, Campylobacter infection is one of the most common cause of acute gastroenteritis, the serious complication for Guillain-Barr and Miller-Fischer syndrome. With the emergence of multi drug resistance is becoming increasingly serious, control and treatment of C. jejuni infection the challenge, the urgent need to control this kind of pathogen infection find new ways. Iron is essential for bacterial growth, although there are very rich in iron, but for pathogenic bacteria, iron bioavailability is very low. For C. jejuni, from the host red cell lysis of hemoglobin heme (heme) is the main source of iron. Play very important role to the mechanism of heme oxygenase ChuZ in the degradation of heme iron and get in The purpose of this study is to analyze the crystal structure of ChuZ, and elucidate the mechanism of C. jejuni utilizing iron from molecular level, so as to provide theoretical basis for developing ChuZ targeted drugs against C. jejuni by blocking the uptake of iron and C. by C. jejuni.
Method:
Cloning of 1.chuZ gene and preparation of recombinant ChuZ protein.
C. jejuni (NCTC11168) genome as template, PCR amplification of chuZ gene and construct the prokaryotic expression vector pET22b. The expression of ChuZ protein by E. coli BL21. In engineering bacteria by affinity chromatography, molecular sieve chromatography and ion exchange chromatography purification method to get high quality ChuZ protein.
2.ChuZ crystallizing condition search.
By using the 72 hole plate Hampton Research company (HR3-086) and crystal screening kit for Index crystal, screen HR110, SaltRx and PEG/ion, were screened preliminary crystallization conditions by Mircobatch method, based on the initial crystallization conditions on the crystallization conditions were optimized to obtain the final crystallization conditions.
3. crystal data collection processing and structural analysis.
The diffraction data of ChuZ-hemin were collected from the X- ray diffraction method. The phase structure of 3GAS was obtained by molecular replacement method. The crystal structure of ChuZ-hemin complex was analyzed and the final model was analyzed.
Analysis of the function of key residues in 4. enzyme activities.
On the basis of structural analysis, preparation of related residues involved in binding mutant substrate heme, the residues involved in binding the substrate heme mutation, enzyme activity was detected and compared with wild-type ChuZ activity, ChuZ enzyme activity test and evaluate the key residues.
5. analysis of the unique surface of the heme binding site.
The binding constants and binding ratios of ChuZ protein and its mutants to heme were measured by UV spectrophotometry and ITC respectively.
Analysis of the crystal structure of 6. mutant.
We prepared the mutant residues related to hemoglobin in combination with hemin. After the combination of ChuZ, we collected the diffraction data and analyzed the crystal structure of the mutant protein by the same method.
Result:
1., a recombinant plasmid pET22b-chuZ expressing ChuZ protein was successfully constructed and expressed in a soluble form. After being combined with various chromatography methods, a high purity ChuZ protein was obtained and the purity of the protein reached over 95%.
2. to optimize the crystal conditions of acicular crystal growth, the final condition is: the concentration of protein 168mg/ml, microbatch in growth conditions: 0.1M MES liquid pool 23%PEG400 50mM pH6.5 iminazole 50mM ammonium tartrate, protein and 1:1 mixed liquid pool after 3 days that the red stick like crystal, seven days after the crystal size is 0.1 x 0.1 x 0.7 mm3.
3. using X- ray diffraction method to collect a set of 2.4? ChuZ-hemin data resolution, the results of data processing: C2221 space group, unit cell parameters a = 106.474, B = 106.698, C = 52.464, alpha beta gamma = =? =90 degrees. Using the PHASER program with HugZ (PDB ID 3GAS) as a model, using molecular replacement method analysis of the 3D structure of ChuZ, the structure of the Split-Barrel folding pattern, structure of monomer heme is significantly different from the classical alpha helix synthase. The key residues involved in substrate binding, such as R166, H9 and H14.
4., R166A, H245Q/R166A, H245Q/H9A/H14A, R166A/H9A/H14A and H9A/H14A mutants were successfully constructed, and R166A/H9A/H14A activity was completely lost by enzyme activity experiments.
5. UV absorbance measurement results show that the ChuZ mutant H9A/H14A with a hemin, wild type ChuZ combined with 1.5 hemin.ITC results showed that the binding constants of H9A/H14A mutant and wild-type ChuZ and other bacteria HO consistent with the proportion of UV and the crystal structure.
6., we obtained the crystal of H9A/H14A mutant and collected a set of 3? Data. Electron density map showed that there was no residual density of heme on the surface of heme binding site, suggesting that the mutant lost the ability to bind heme.
Conclusion:
The three-dimensional structure of ChuZ Split-Barrel by folding the unique way, structure of the monomer heme and obtain the most alpha helix analytical synthase is obviously different, belong to a to HugZ as the representative of the new family of heme oxygenase.ChuZ in solution and in the crystal as Homo dimer has two fine structure in ChuZ. The discovery of a new heme binding site, and it exists in the heme protein in "classic sites", four histidine on the surface of the enzyme by water molecule mediated binding to the heme binding mode, like in other heme oxygenase has not been found. The study demonstrated that ChuZ is a new kind of the heme binding protein family members special mutagenesis, and through the analysis of the relationship between structure and function of ChuZ and homologous protein comparative study, found that heme C domain binding pocket "" is the key structural basis of the ChuZ enzyme catalysis mechanism.

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

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