Elahi Montasir, Islam Monirul M, Noguchi Keiichi, Yohda Masafumi, Toh Hiroyuki, Kuroda Yutaka
Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture & Technology, Nakamachi, Koganei-shi, Tokyo 184-8588, Japan.
Instrumentation Analysis Center, Tokyo University of Agriculture & Technology, Nakamachi, Koganei-shi, Tokyo 184-8588, Japan.
Biochim Biophys Acta. 2014 Mar;1844(3):585-92. doi: 10.1016/j.bbapap.2013.12.013. Epub 2013 Dec 27.
Dengue viruses (DEN) are classified into four serotypes (DEN1-DEN4) exhibiting high sequence and structural similarities, and infections by multiple serotypes can lead to the deadly dengue hemorrhagic fever. Here, we aim at characterizing the thermodynamic stability of DEN envelope protein domain III (ED3) during its evolution, and we report a structural analysis of DEN4wt ED3 combined with a systematic mutational analysis of residues 310 and 387. Molecular modeling based on our DEN3 and DEN4 ED3 structures indicated that the side-chains of residues 310/387, which are Val(310)/Ile(387) and Met(310)/Leu(387) in DEN3wt and DEN4wt, respectively, could be structurally compensated, and that a "size switch type repacking" might have occurred at these sites during the evolution of DEN into its four serotypes. This was experimentally confirmed by a 10°C and 5°C decrease in the thermal stability of, respectively, DEN3 ED3 variants with Met(310)/Ile(387) and Val(310)/Leu(387), whereas the variant with Met(310)/Leu(387), which contains a double mutation, had the same stability as the wild type DEN3. Namely, the Met310Val mutation should have preceded the Leu387Ile mutation in order to maintain the tight internal packing of ED3 and thus its thermodynamic stability. This view was confirmed by a phylogenetic reconstruction indicating that a common DEN ancestor would have Met(310)/Leu(387), and the intermediate node protein, Val(310)/Leu(387), which then mutated to the Val(310)/Ile(387) pair found in the present DEN3. The hypothesis was further confirmed by the observation that all of the present DEN viruses exhibit only stabilizing amino acid pairs at the 310/387 sites.
登革病毒（DEN）分为四种血清型（DEN1 - DEN4），它们在序列和结构上具有高度相似性，多种血清型感染可导致致命的登革出血热。在此，我们旨在表征登革病毒包膜蛋白结构域III（ED3）在其进化过程中的热力学稳定性，并报告DEN4wt ED3的结构分析以及对310和387位残基的系统突变分析。基于我们的DEN3和DEN4 ED3结构进行的分子建模表明，310/387位残基的侧链，在DEN3wt和DEN4wt中分别为Val(310)/Ile(387)和Met(310)/Leu(387)，在结构上可以相互补偿，并且在登革病毒进化为四种血清型的过程中，这些位点可能发生了“尺寸开关型重排”。这通过实验得到了证实，分别将Met(310)/Ile(387)和Val(310)/Leu(387)引入DEN3 ED3变体后，其热稳定性分别降低了10°C和5°C，而含有双突变的Met(310)/Leu(387)变体与野生型DEN3具有相同的稳定性。也就是说，为了维持ED3紧密的内部堆积从而保持其热力学稳定性，Met310Val突变应该先于Leu387Ile突变发生。系统发育重建证实了这一观点，表明登革病毒的共同祖先具有Met(310)/Leu(387)，中间节点蛋白具有Val(310)/Leu(38)，然后突变为当前DEN3中发现的Val(310)/Ile(387)对。所有当前的登革病毒在310/387位点仅表现出稳定的氨基酸对这一观察结果进一步证实了该假设。
