Lin Thy-Hou, Tsai Keng-Chang, Lo Ta-Chun
Department of Life Science, National Tsing Hua University, Hsinchu 30043, Taiwan.
Protein Eng. 2003 Nov;16(11):819-29. doi: 10.1093/protein/gzg107.
The tertiary structure of the central catalytic domain of insertion sequence ISLC3 isolated from Lactobacillus casei ATCC 393 was predicted using the homology modeling approach. The novel insertion sequence was isolated by us from the template bacteriophage phiA3 of L.casei ATCC 393. The number of amino acid residues of the ISLC3 central catalytic domain was 116 and was treated as the query sequence. There were five Web-available threading methods used to find some primary structure templates for the query sequence. These primary templates were further screened using the SWISS-MODEL Protein Modeling Server and the default parameter settings therein to give six final structure templates. All of these final structure templates were the integrase (IN) protein of retroviruses. Multiple sequence alignment using these IN sequences against the query one revealed the signature DDE motif. Based on the structures of these final templates, the structure of the query sequence was constructed using the InsightII/Discover/Homology programs. A metal ion, Mg(2+), was inserted into the center of the putative catalytic pocket formed by the DDE residues of the predicted structure in the final rounds of refinement by molecular dynamics (MD) simulations. The structure with a metal ion included was designated with Mg and that without a metal ion was designated free Mg. The average exposed surface area of some hydrophobic residues of both the predicted free Mg and with Mg structures were computed and compared with those computed for the six structure templates. Whereas the predicted with Mg structure was slightly more exposed than the predicted free Mg structure, the former appeared to be more stable than the latter, as revealed by the lower conformation energy recorded for the former during the structure refinement by MD simulations. To verify further the predicted structures, the coordinates of both predicted structures were fed into the ERRAT Protein Verification Server. It was found that the quality of the predicted with Mg structure was much better than that of the free Mg structure. The validation results also indicated that regions of the predicted with Mg structure that can be rejected at the 95% confidence level were approximately 20% whereas those which can be rejected at the same level for the six structure templates were approximately 10%. The predicted with Mg structure was also docked into a short oligonucleotide representing the substrate of the ISLC3 transposase using the DOCK_4.0.2 program. It was found that both Glu140 and Asp68 residues of the DDE motif of the predicted with Mg structure were able to form hydrogen bonds with the DNA substrate, which was similar to what was observed in a docking study using the retrovirus IN 1asu and its DNA substrate.
采用同源建模方法预测了从干酪乳杆菌ATCC 393中分离出的插入序列ISLC3中央催化结构域的三级结构。我们从干酪乳杆菌ATCC 393的模板噬菌体phiA3中分离出了这种新型插入序列。ISLC3中央催化结构域的氨基酸残基数为116，并将其作为查询序列。使用了五种可在网络上获取的穿线法来为查询序列寻找一些一级结构模板。这些一级模板通过SWISS-MODEL蛋白质建模服务器及其默认参数设置进行进一步筛选，得到六个最终结构模板。所有这些最终结构模板均为逆转录病毒的整合酶（IN）蛋白。使用这些IN序列与查询序列进行多序列比对，揭示了特征性的DDE基序。基于这些最终模板的结构，使用InsightII/Discover/Homology程序构建查询序列的结构。在分子动力学（MD）模拟的最后一轮优化中，将金属离子Mg(2+)插入到由预测结构的DDE残基形成的假定催化口袋中心。包含金属离子的结构标记为Mg，不包含金属离子的结构标记为游离Mg。计算了预测的游离Mg结构和含Mg结构中一些疏水残基的平均暴露表面积，并与六个结构模板计算得到的结果进行比较。虽然预测的含Mg结构比预测的游离Mg结构略微更暴露，但如MD模拟结构优化过程中前者记录的较低构象能所示，前者似乎比后者更稳定。为了进一步验证预测的结构，将两种预测结构的坐标输入ERRAT蛋白质验证服务器。发现含Mg结构的预测质量比游离Mg结构好得多。验证结果还表明，含Mg结构中在95%置信水平下可被拒绝的区域约为20%，而六个结构模板在相同水平下可被拒绝的区域约为10%。使用DOCK_4.0.2程序将含Mg结构的预测结果对接至代表ISLC3转座酶底物的短寡核苷酸。发现含Mg结构预测的DDE基序中的Glu140和Asp68残基均能够与DNA底物形成氢键，这与使用逆转录病毒IN 1asu及其DNA底物的对接研究中观察到的情况相似。
