【作者】 童水龙；

【导师】 牛立文； 滕脉坤；

【作者基本信息】 中国科学技术大学 ， 结构生物学， 2008， 博士

【摘要】 (Ⅰ)破骨细胞是负责骨吸收的主要细胞。它的分化和成熟需要M-CSF（macrophage colony stimulating factor）和RANKL（receptor activator ofnuclear factor kappaB ligand）介导的信号通路网络。酪氨酸激酶Src,它参与了M-CSF和RANKL介导的信号网络,对破骨细胞的功能的发挥有重要作用。破骨细胞刺激因子（OSF）是由一个短的富含脯氨酸区域,一个SH3结构域和ankyrinrepeats组成的胞内蛋白,它可以间接地刺激破骨细胞的形成和活化。OSF在鼠中的同源蛋白SH3P2可以结合Cbl上的一段proline rich的区域并形成OSF-Cbl-Src的三蛋白复合物,说明它参与了Src和Cbl介导的信号通路。我们克隆、表达并纯化了人的OSF蛋白,并进行了晶体生长,在同一条件下获得了两种空间群的晶体,并解出了它们的结构。第一种晶型我们用多波长反常散射的方法解出了它的2.57（?）的晶体结构,它属于P2₁2₁2₁空间群,晶胞参数为a=48.06 （?）,b=56.86 （?）和c=170.12 （?）,最后修正的R因子和Rfree因子分别为21.0%和28.4%。第二种晶型的结构使用分子置换法解得了1.95（?）得结构,它属于P1空间群,晶胞参数为a=28.31（?）,b=57.94 （?）,c=60.48 （?）,α=61.75°,β=76.53°,γ=90.08°,最后修正的R因子和Rfree因子分别为17.8%和23.8%。在最后的模型中,两种晶型都含有SH3结构域和ankyrin repeats结构域,只是结构域之间的相对位置有变化。这是第一个报道的具有SH3-ANKs这样的结构域排布顺序的结构。我们发现OSF-SH3结构域尾部的配体结合区（螺旋区域）明显与其它的SH3结构域不同,暗示它与proline rich配体之间的相互作用很可能需要构象的改变,并且这个构象的变化可能是受到后面的ankyrin repeats结构域的调控的。（Ⅱ）来源于普通变形杆菌的L-氨基酸脱氨酶（LAD）可以催化多种L-氨基酸脱氨。在LAD的序列中发现存在保守的FAD结合区域,所以LAD可能是属于L-氨基酸氧化酶（LAAO）。我们构建了LAD的全长和氨基端29个残基缺失的△N29LAD质粒,表达并纯化了这两种LAD,并进行了晶体生长。我们得到了得到了△N29LAD适合衍射的晶体。我们收集了△N29LAD的一套2.9 （?）的数据,并进行了初步晶体学分析。更多还原

【Abstract】 (Ⅰ) Osteoclast is the main cell responsible for degradation of bone matrix. Its differentiation and activity requires M-CSF （macrophage colony stimulating factor） and RANKL （receptor activator of nuclear factor kappaB ligand） mediated pathways. Tyrosine protein kinase Src, involved in both M-CSF and RANKL mediated signal networks, is also found to be essential for osteoclast function. Osteoclast stimulating factor （OSF）, composed of a short proline rich region, one SH3 domain and ankyrin repeats, is a intracellular protein produced by osteoclasts and shown to indirectly enhance osteoclast formation and activity. A mouse homolog of OSF called SH3P2 can bind proline-rich fragment of Cbl and form an OSF-Cbl-Src triple protein complex, suggesting OSF was involved in Src and Cbl mediated pathways.We cloned, expressed and purified the human OSF protein. Here, we present crystal structures of human OSF in two space groups grown from the same condition. Form I crystal structure is determined by multi-wavelength anomalous diffraction （MAD） at 2.57A resolution in space group P2₁2₁2₁ with cell parameters a=48.06 （?）, b=56.86 （?） and c=l70.12 （?）.The final R value and Rfree value are 21.0% and 28.4%, respectively. FormⅠcrystal structure is determined by molecular replacement at 1.95 （?） resolution in space group P1 with cell parameters a=28.31（?）, b=57.94 （?）, c=60.48（?）,α=61.75°,β=76.53°,γ=90.08°.The final R value and Rfree value are 17.8% and 23.8%, respectively. Models in both crystal forms contain one SH3 domain and four ankyrin repeats, but the relative position of the two domains is different in two forms. It is the first reported protein structure of such a SH3-ANKs domain sequence. The peptide-binding groove of OSF-SH3 domain adopts a conformation different from other SH3 domains and probably needs a conformational switch for binding to its proline rich ligand. This different status of OSF-SH3 is possibly regulated by the C-terminal ankyrin repeats. （Ⅱ）L-amino acid deaminase（LAD）from Proteus vulgaris is an enzyme that cancatalyze L-type amino acids.Significant conserved motif is found in LAD identifiedas a FAD-binding domain and probably LAD is a L-amino acid oxidase．We cloned，expressed and purified the full length LAD and a N-terminal truncation form△N29LAD.Crystals of△N29LAD suitable for diffraction were obtained and a 2.9 （?）data set was Collected.Preliminary X-ray diffraction analysis was applied.更多还原