Signature Active Site Architectures Illuminate the Molecular Basis for Ligand Specificity in Family 35 Carbohydrate Binding Module
Signature Active Site Architectures Illuminate the Molecular Basis for Ligand Specificity in Family 35 Carbohydrate Binding Module
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DOI：
10.1021/bi1006139
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发表时间：
2010-07-27
期刊：
影响因子：
2.9
通讯作者：
Gilbert, Harry J.
中科院分区：
文献类型：
--
作者：
Correia, Marcia A. S.;Abbott, D. Wade;Gilbert, Harry J.
植物细胞壁的解构是一个重要的生物过程，正在吸引相当大的工业兴趣，特别是在生物能源部门。攻击植物细胞壁的酶通常含有一个或多个非催化碳水化合物结合模块（CBMs），这些模块起着重要的靶向作用。虽然与植物结构多糖骨架结合的CBMs已经被广泛描述，但识别这些聚合物上显示的大量装饰成分的模块相对来说尚未被探索。在这里，我们展示了一个家族35 CBM成员（CBM35），被命名为CtCBM35-Gal，与a- d -半乳糖（Gal）结合，并且在植物细胞壁的背景下，靶向半乳甘露聚糖的α -1,6-Gal残基，而不是木葡聚糖的β - d -Gal残基。CtCBM35-Gal的晶体结构呈现典型的β -三明治折叠。定点诱变研究表明，配体被安置在连接两个β -片的环内。尽管CBM的配体结合位点与靶向糖醛酸的钙依赖性CBM35s具有显著的结构相似性，但配体结合位点上保守残基构象的细微差异导致金属结合和糖醛酸识别的丧失。提出了一个模型，其中与Gal吡喃糖环的两个面相互作用的对芳香残基的取向对配体的轴向O4原子朝向Asn140起关键作用，这在CBM35中是不变的。外显子CBM35s的配体识别位点（CBM35-Gal和结合CBM35s的醛酸）似乎与CBM35-Man的配体识别位点重叠，后者结合甘露糖的β -聚合物甘露糖的内部区域。通过位点定向诱变，我们发现尽管在CBM35和CBM35的外显子结合位点中存在一些功能残基的保守性，但内显子cbm在甘露聚糖结合中不利用Asn113（相当于CBM35- gal中的Asn140），尽管等效残基在CBM35和CBM6的配体识别中很重要。本报告中提供的数据是在CBM35家族更广泛的系统发育背景下进行的。
The deconstruction of the plant cell wall is an important biological process that is attracting considerable industrial interest, particularly in the bioenergy sector. Enzymes that attack the plant cell wall generally contain one or more noncatalytic carbohydrate binding modules (CBMs) that play an important targeting function. While CBMs that bind to the backbones of plant structural polysaccharides have been widely described, modules that recognize components of the vast array of decorations displayed on these polymers have been relatively unexplored. Here we show that a family 35 CBM member (CBM35), designated CtCBM35-Gal, binds to a-D-galactose (Gal) and, within the context of the plant cell wall, targets the alpha-1,6-Gal residues of galactomannan but not the beta-D-Gal residues in xyloglucan. The crystal structure of CtCBM35-Gal reveals a canonical beta-sandwich fold. Site-directed mutagenesis studies showed that the ligand is accommodated within the loops that connect the two beta-sheets. Although the ligand binding site of the CBM displays significant structural similarity with calcium-dependent CBM35s that target uronic acids, subtle differences in the conformation of conserved residues in the ligand binding site lead to the loss of metal binding and uronate recognition. A model is proposed in which the orientation of the pair of aromatic residues that interact with the two faces of the Gal pyranose ring plays a pivotal role in orientating the axial O4 atom of the ligand toward Asn140, which is invariant in CBM35. The ligand recognition site of exo-CBM35s (CBM35-Gal and the uronic acid binding CBM35s) appears to overlap with that of CBM35-Man, which binds to the internal regions of mannan, a beta-polymer of mannose. Using site-directed mutagenesis, we show that although there is conservation of several functional residues within the binding sites of endo- and exo-CBM35s, the endo-CBM does not utilize Asn113 (equivalent to Asn140 in CBM35-Gal) in mannan binding, despite the importance of the equivalent residue in ligand recognition across the CBM35 and CBM6 landscape. The data presented in this report are placed within a wider phylogenetic context for the CBM35 family.