Structural Studies On Mycobacterium Smegmatis Dps Molecules
Siddhartha Roy · 2007
Oxidative stress is a universal phenomenon experienced by both aerobic and anaerobic organisms. Reactive oxygen species (ROS) are generated during the stress, which can damage most cellular components including proteins, lipids and DNA. Naturally, organisms have evolved defence mechanisms to prevent oxidative damage. In prokaryotic systems, Dps (DNA binding protein from stationary phase cells) forms an important component of the mechanisms. Dps is known to be produced maximally during the stationary phase of bacterial growth. They exhibit ferroxidase activity as well. Dps homologs have been identified in a variety of distantly related bacteria, thus implying that this protein has a crucial function. The crystal structures of these proteins from a few bacteria are available. The work reported here is concerned with structural studies on Dps molecules from Mycobacterium smegmatis. Well-established X-ray crystallographic techniques were used to study the structures reported here. Hanging drop vapour diffusion and microbatch methods were used for crystallization. X-ray intensity data were collected on MAR Research imaging plates mounted on Rigaku X-ray generators. The data were processed using the HKL program suite. All the structures were solved by the molecular replacement method using the programs AMoRe and PHASER. Structure refinements were carried out using the programs CNS and REFMAC. Model building was carried out using FRODO and COOT. PROCHECK, ALIGN, INSIGHT, NACCESS, HBPLUS, CONTACT and ESCET were used for validation and analysis of the refined structures. Figures were prepared using MOLSCRIPT, BOBSCRIPT, RASTER3D and PYMOL. The structure of the first Dps identified in M. smegmatis has been determined in three crystal forms and has been compared with those of similar proteins from other sources. The dodecameric molecule can be described as a distorted icosahedron. The interfaces among subunits are such that the dodecameric molecule appears to have been made up of stable trimers. The situation is similar in the proteins from Escherichia coli and Agrobacterium tumefaciens, which are closer to the M. smegmatis protein in sequence and structure than those from other sources, which appear to form a dimer first. Trimerisation is aided in the three proteins by the additional N-terminal stretches they possess. The M. smegmatis protein has an additional C-terminal stretch compared to other related proteins. The stretch, known to be involved in DNA binding, is situated on the surface of the molecule. A comparison of the available structures permits a delineation of the rigid and flexible regions in the molecule. The subunit interfaces around the molecular dyads, where the ferroxidation centres are located, are relatively rigid. Regions in the vicinity of the acidic holes centred around molecular threefold axes, are relatively flexible. So are the DNA binding regions. The crystal structures of the protein from M. smegmatis confirm that DNA molecules can occupy spaces within the crystal without disturbing the…