Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases.
Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases.
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通过配体特异性洗脱从染料树脂中分析核苷结合蛋白：应用于结核分枝杆菌醛醛脱氢酶。
DOI：
10.1007/s10969-009-9073-z
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发表时间：
2009-12
期刊：
影响因子：
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通讯作者：
Terwilliger, Thomas C
中科院分区：
文献类型：
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作者：
Kim, Chang-Yub;Webster, Cecelia;Roberts, Justin K M;Moon, Jin Ho;Alipio Lyon, Emily Z;Kim, Heungbok;Yu, Minmin;Hung, Li-Wei;Terwilliger, Thomas C
我们表明，Cibacron蓝F3 GA染料树脂色谱法可用于识别配体，特异性地与结核分枝杆菌的蛋白质相互作用，这些配体的识别可以通过提高晶体的质量来促进结构测定。醛脱氢酶（ALDH）家族的四种天然Mtb蛋白先前显示出从具有核苷的汽巴克隆蓝F3 GA染料树脂特异性洗脱。在这项研究中，我们的特点之一，这些ALDH同工酶（重组结核分枝杆菌Rv 0223 c）的核苷结合特异性，并比较这些生化结果与不同的Rv 0223 c-核苷配对的共结晶实验。我们发现，强相互作用的配体（NAD和NADH）有助于形成高质量的晶体，允许第一个Mtb ALDH（Rv 0223 c）结构的解决方案。其他核苷配体（AMP、FAD、腺苷、GTP和NADP）表现出与Rv 0223 c的较弱结合，并产生衍射至较低分辨率的共晶体。基于Rv 0223 c与各种核苷配体的晶体的差异电子密度图显示大多数共享天然配体NAD结合的结合位点。从与人线粒体ALDH-2相比的序列和结构的高度相似性（BLAST Z-评分= 53.5和RMSD = 1.5分）来看，Rv 0223 c似乎属于ALDH-2类别。Rv 0223 c结构中改变的寡聚化结构域似乎将该蛋白保持为单体，而天然人ALDH-2是多聚体。本文的在线版本（doi：10.1007/s10969-009-9073-z）包含补充材料，可供授权用户使用。
We show that Cibacron Blue F3GA dye resin chromatography can be used to identify ligands that specifically interact with proteins from Mycobacterium tuberculosis, and that the identification of these ligands can facilitate structure determination by enhancing the quality of crystals. Four native Mtb proteins of the aldehyde dehydrogenase (ALDH) family were previously shown to be specifically eluted from a Cibacron Blue F3GA dye resin with nucleosides. In this study we characterized the nucleoside-binding specificity of one of these ALDH isozymes (recombinant Mtb Rv0223c) and compared these biochemical results with co-crystallization experiments with different Rv0223c-nucleoside pairings. We found that the strongly interacting ligands (NAD and NADH) aided formation of high-quality crystals, permitting solution of the first Mtb ALDH (Rv0223c) structure. Other nucleoside ligands (AMP, FAD, adenosine, GTP and NADP) exhibited weaker binding to Rv0223c, and produced co-crystals diffracting to lower resolution. Difference electron density maps based on crystals of Rv0223c with various nucleoside ligands show most share the binding site where the natural ligand NAD binds. From the high degree of similarity of sequence and structure compared to human mitochondrial ALDH-2 (BLAST Z-score = 53.5 and RMSD = 1.5 Å), Rv0223c appears to belong to the ALDH-2 class. An altered oligomerization domain in the Rv0223c structure seems to keep this protein as monomer whereas native human ALDH-2 is a multimer. The online version of this article (doi:10.1007/s10969-009-9073-z) contains supplementary material, which is available to authorized users.