Sujatha M S, Balaji Petety V
School of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Powai, Mumbai 400 076, India.
BMC Struct Biol. 2006 Apr 19;6:9. doi: 10.1186/1472-6807-6-9.
The 3-D structure of none of the eukaryotic sialyltransferases (SiaTs) has been determined so far. Sequence alignment algorithms such as BLAST and PSI-BLAST could not detect a homolog of these enzymes from the protein databank. SiaTs, thus, belong to the hard/medium target category in the CASP experiments. The objective of the current work is to model the 3-D structures of human SiaTs which transfer the sialic acid in alpha2,3-linkage viz., ST3Gal I, II, III, IV, V, and VI, using fold-recognition and comparative modeling methods. The pair-wise sequence similarity among these six enzymes ranges from 41 to 63%.
Unlike the sequence similarity servers, fold-recognition servers identified CstII, a alpha2,3/8 dual-activity SiaT from Campylobacter jejuni as the homolog of all the six ST3Gals; the level of sequence similarity between CstII and ST3Gals is only 15-20% and the similarity is restricted to well-characterized motif regions of ST3Gals. Deriving template-target sequence alignments for the entire ST3Gal sequence was not straightforward: the fold-recognition servers could not find a template for the region preceding the L-motif and that between the L- and S-motifs. Multiple structural templates were identified to model these regions and template identification-modeling-evaluation had to be performed iteratively to choose the most appropriate templates. The modeled structures have acceptable stereochemical properties and are also able to provide qualitative rationalizations for some of the site-directed mutagenesis results reported in literature. Apart from the predicted models, an unexpected but valuable finding from this study is the sequential and structural relatedness of family GT42 and family GT29 SiaTs.
The modeled 3-D structures can be used for docking and other modeling studies and for the rational identification of residues to be mutated to impart desired properties such as altered stability, substrate specificity, etc. Several studies in literature have focused on the development of tools and/or servers for the large-scale/automated modeling of 3-D structures of proteins. In contrast, the present study focuses on modeling the 3-D structure of a specific protein of interest to a biochemist and illustrates the associated difficulties. It is also able to establish a sequence/structure relationship between sialyltransferases of two distinct families.
迄今为止，尚无真核生物唾液酸转移酶（SiaTs）的三维结构被确定。诸如BLAST和PSI-BLAST等序列比对算法无法从蛋白质数据库中检测到这些酶的同源物。因此，在蛋白质结构预测技术关键评估（CASP）实验中，SiaTs属于难/中等难度的目标范畴。当前工作的目标是使用折叠识别和比较建模方法，对在α2,3-连接中转移唾液酸的人类SiaTs（即ST3Gal I、II、III、IV、V和VI）的三维结构进行建模。这六种酶之间的成对序列相似性范围为41%至63%。
与序列相似性服务器不同，折叠识别服务器将空肠弯曲杆菌的一种α2,3/8双活性SiaT即CstII识别为所有六种ST3Gal的同源物；CstII与ST3Gal之间的序列相似性水平仅为15%-20%，且这种相似性仅限于ST3Gal特征明确的基序区域。为整个ST3Gal序列推导模板-靶序列比对并非易事：折叠识别服务器无法为L基序之前的区域以及L基序和S基序之间的区域找到模板。已识别出多个结构模板来对这些区域进行建模，并且必须迭代地进行模板识别-建模-评估以选择最合适的模板。所建模的结构具有可接受的立体化学性质，并且还能够为文献中报道的一些定点诱变结果提供定性的合理解释。除了预测模型之外，本研究一个意外但有价值的发现是GT42家族和GT29家族SiaTs的序列和结构相关性。
所建模的三维结构可用于对接和其他建模研究，以及合理确定要突变的残基，以赋予所需特性，如改变稳定性、底物特异性等。文献中的几项研究集中于开发用于蛋白质三维结构大规模/自动化建模的工具和/或服务器。相比之下；本研究专注于对生物化学家感兴趣的特定蛋白质的三维结构进行建模，并阐述了相关的困难。它还能够在两个不同家族的唾液酸转移酶之间建立序列/结构关系。
