Crystallographic And Structural Analysis Of Streptomyces Coelicolor Calcium-binding Protein

Calcium ions plays a significant role in a myriad of cellular physiological processes, including signal transduction, cell cycle regulation, neurotransmitters release, glycogen metabolism, and apoptosis. In eukaryotes, the important and diverse regulatory roles of Ca2+ are conveyed by the highly conserved and ubiquitous calmodulin superfamily. However, the calcium-regulatory proteins in prokaryotes are still hypothetical or poorly understood. Streptomyces coelicolor is a representative of the bacteria responsible for producing most natural antibiotics used in human and veterinary medicine. The study of the protein structure presumably involved in the secondary metabolism and the assumed regulatory mechanism can provide a structural basis for improving the secondary metabolic products as well as developing drugs.Here we report the expression, purification, crystallization, and structure determination and analysis of Streptomyces coelicolor calcium-binding protein using S. coelicolor as the platform. The recombinant proteins were highly expressed in Escherichia coli and purified; well-diffracting crystals were obtained by hanging-drop vapor diffusion method. The crystals belong to a space group P212121 and diffract to 1.5? resolution on a synchrotron source with unit cell parameters a=33.1?, b=51.1?, c=87.3?,α=β=γ=90°. The crystal structure of CabD has been solved by singlewavelegth anomalous diffraction method. The three-dimensional structure of CabD is a compact globular molecule with eightα-helical segments. Two neighboringα-helices and the connecting loop compose the helix-loop-helix EF-hand motif. EF-hand pairs separated on opposite sides of the molecule are linked by a kink consisting of only one residue Lys92, and are arranged to form a pronounced hydrophobic core comprising about 20 aromatic residues.In order to better understand the potentially calcium-mediated biological role of CabD, we superimpose the three-dimensional structure of CabD with the related four EF-hand motif containing calcium-binding proteins. The lack of the extreme C-terminal loop beyond the last EF-hand of CabD makes the opening of hydrophobic pocket more accessible to the putative target recognition or binding. The study of the tertiary structure of the Streptomyces coelicolor calcium-binding protein CabD may give an insight into the calcium signal transduction in prokaryotes provided with a tremendously theoretical significance and economic benefits.