A. Rendón-Ramírez, J. Agirre, A. E. Mechaly, A. and D. M. A. Guérin
Unidad de Biofísica (CSIC–UPV/EHU),Fundación Biofísica Bizkaia, PO Box 644, E-48080
The prokaryotic K+ channel KcsA-sl (from Streptomyces lividans), is analogous to eukaryotic K+ channels. KcsA has been recently crystallized usingmonoclonal antibodies raised against the middle part of the long extracellular domain (Marien D., et all 2001). A crystallization chaperone is an auxiliary protein that binds to a target of interest,enhances and modulates crystal packing, and can even provide useful phasing information. Calmodulin (CaM) was found to promiscuously bind to the intracellular region of several eukaryotic potassiumchannels (Schumacher et al., 2001). Therefore, we envisage using recombinant rat brain CaM as a crystallization chaperone of KcsA-sl.
In this work we report a preliminary study using differenttechniques that account for interactions between the two proteins. First, using pull-down experiments, we found a direct interaction between KcsA and CaM-Ca2+. Second, the fluorescence of dansyl-calmodulin(CaMd) in the presence of KcsA was increased in 100 uM free Ca2+ compared to 0 uM free Ca2+. And third, NMR spectroscopy reveals that the N-terminal side of the effector domain has interaction withthe C-terminal domain of KcsA, whereas the C-terminal side of the effector domain contacts the N-terminal domain of CaM.
Finally, the change in the CaMd fluorescent spectrum, being alone ormixed with KcsA, clearly reveals an interaction between both proteins. We propose that KcsA binds to CaM and induces it conformational rearrangements. Furthermore, it is interesting from an evolutionarypoint of view that a protein that is expressed exclusively in eukaryotic cells can bind to a prokaryotic channel. We are currently running crystallization experiments without noticeable results....