Edema factor from Bacillus anthracis is a 92 kDa secreted adenylyl cyclase exotoxin and is activated by the host-resident protein calmodulin. Calmodulin is a ubiquitous intracellular calcium sensor in eukaryotes and activates edema factor nearly 1000-fold upon binding. While calmodulin has many known effectors, including kinases, phosphodiesterases, motor proteins, channels and type 1 adenylyl cyclases, no structures of calmodulin in complex with a functional enzyme have been solved. The crystallization and initial experimental phasing of crystals containing a complex of edema factor adenylyl cyclase domain and calmodulin are reported here. The edema factor–calmodulin complex crystallizes in three different space groups. A native data set in the I222 space group has been collected to 2.7 Å and the self-rotation function solution suggests three edema factor–calmodulin complexes in each asymmetric unit. Initial 4 Å phases were obtained by selenomethionyl MAD in combination with two heavy-atom derivatives. These phases were successfully extended to 2.7 Å using NCS averaging.
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Gene regulation depends on proteins that bind to specific DNA sites. Such specific recognition often involves severe DNA deformations, including sharp kinks. It has been unclear how rigid or flexible these protein-induced kinks are. Here, we investigated the dynamic nature of DNA in complex with integration host factor (IHF), a nucleoid-associated architectural protein known to bend one of its cognate sites (35 base pair H′) into a U-turn by kinking DNA at two sites. We utilized fluorescence-lifetime-based FRET spectroscopy to assess the distribution of bent conformations in various IHF–DNA complexes. Our results reveal a surprisingly dynamic specific complex: while 78% of the IHF–H′ population exhibited FRET efficiency consistent with the crystal structure, 22% exhibited FRET efficiency indicative of unbent or partially bent DNA. This conformational flexibility is modulated by sequence variations in the cognate site. In another site (H1) that lacks the A-tract of H′ found on one side of the binding site, the extent of bending in the fully U-bent conformation decreased, and the population in that state decreased to 32%. A similar decrease in the U-bent population was observed with a single base mutation in H′ in a consensus region on the other side. Taken together, these results provide important insights into the finely tuned interactions between IHF and its cognate sites that keep the DNA bent (or not) and yield quantitative data on the dynamic equilibrium between different DNA conformations (kinked or not kinked) that depend sensitively on DNA sequence and deformability. Notably, the difference in dynamics between IHF–H′ and IHF–H1 reflects the different roles of these complexes in their natural context, in the phage lambda "intasome" (the complex that integrates phage lambda into the E. coli chromosome).
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