Roy Siddhartha, Saraswathi Ramachandran, Gupta Surbhi, Sekar K, Chatterji Dipankar, Vijayan M
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
J Mol Biol. 2007 Jul 20;370(4):752-67. doi: 10.1016/j.jmb.2007.05.004. Epub 2007 May 10.
Mycobacterium smegmatis Dps degrades spontaneously into a species in which 16 C-terminal residues are cleaved away. A second species, in which all 26 residues constituting the tail were deleted, was cloned, expressed and purified. The first did not bind DNA but formed dodecamers like the native protein, while the second did not bind to DNA and failed to assemble into dodecamers, indicating a role in assembly also for the tail. In the crystal structure of the species without the entire C-terminal tail the molecule has an unusual open decameric structure resulting from the removal of two adjacent subunits from the original dodecameric structure of the native form. A Dps dodecamer could assemble with a dimer or one of two trimers (trimer-A and trimer-B) as intermediate. Trimer-A is the intermediate species in the M. smegmatis protein. Estimation of the surface area buried on trimerization indicates that association within trimer-B is weak. It weakens further when the C-terminal tail is removed, leading to the disruption of the dodecameric structure. Thus, the C-terminal tail has a dual role, one in DNA binding and the other in the assembly of the dodecamer. M. smegmatis Dps also has a short N-terminal tail. A species with nine N-terminal residues deleted formed trimers but not dodecamers in solution, unlike wild-type M. smegmatis Dps, under the same conditions. Unlike in solution, the N-terminal mutant forms dodecamers in the crystal. In native Dps, the N-terminal stretch of one subunit and the C-terminal stretch of a neighboring subunit lock each other into ordered positions. The deletion of one stretch results in the disorder of the other. This disorder appears to result in the formation of a trimeric species of the N-terminal deletion mutant contrary to the indication provided by the native structure. The ferroxidation site is intact in the mutants.
耻垢分枝杆菌Dps会自发降解为一种C末端16个残基被切除的物种。克隆、表达并纯化了第二种物种，其中构成尾部的所有26个残基均被删除。第一种物种不结合DNA，但像天然蛋白质一样形成十二聚体，而第二种物种不与DNA结合，也无法组装成十二聚体，这表明尾部在组装中也起作用。在没有完整C末端尾部的物种的晶体结构中，分子具有不寻常的开放十聚体结构，这是由于从天然形式的原始十二聚体结构中去除了两个相邻亚基所致。一个Dps十二聚体可以与一个二聚体或两个三聚体（三聚体A和三聚体B）之一作为中间体组装。三聚体A是耻垢分枝杆菌蛋白中的中间物种。三聚化时埋藏表面积的估计表明三聚体B内的缔合较弱。当C末端尾部被去除时，它会进一步减弱，导致十二聚体结构的破坏。因此，C末端尾部具有双重作用，一个在DNA结合中，另一个在十二聚体的组装中。耻垢分枝杆菌Dps也有一个短的N末端尾部。与野生型耻垢分枝杆菌Dps不同，在相同条件下，一种缺失九个N末端残基的物种在溶液中形成三聚体但不形成十二聚体。与在溶液中不同，N末端突变体在晶体中形成十二聚体。在天然Dps中，一个亚基的N末端延伸和相邻亚基的C末端延伸相互锁定在有序位置。一个延伸的缺失导致另一个的无序。这种无序似乎导致N末端缺失突变体形成三聚体物种，这与天然结构提供的指示相反。铁氧化位点在突变体中是完整的。
