Conformational Changes in the Ca -regulatory Region from Soybean Calcium-dependent Protein Kinase-

Calcium-dependent protein kinases are key proteins involved in plant and protozoal Ca signaling. These unique molecules include a calcium regulatory calmodulin-like domain (CLD), which binds to another small regulatory domain named the junction domain (JD). Both CLD and JD are part of the same polypeptide as the protein kinase domain. The CLD consists of Nand C-terminal lobes, each having two helix-loop-helix Ca -binding motifs. In this study, fluorescence resonance energy transfer using a series of Trp and Cys site-directed mutants was undertaken to probe the relative motions of the two lobes of CLD between the apoand Ca -saturated forms, as well as bound to a peptide encoding the JD sequence. Using an IAEDANS-modified Cys, a total of 15 Trp3 Cys distances were measured in these three states from the five donor-acceptor combinations F334W-Cys, L371W-Cys, L403W-Cys, F334W-L403C, and L371WL403C. Consistent with recently reported NMR diffusion measurements and with H,N heteronuclear correlation NMR spectra, the distances derived from fluorescence resonance energy transfer measurements in apoCLD indicate partial unfolding and a subsequent contraction on binding Ca , which is maintained on addition of the JD peptide. Interpretation of the distances suggests that the Ca -saturated form is open with the two lobes sitting side-by-side although highly flexible. The transition to the JD-CLD state appears to be accompanied by a rotation of the Nand C-terminal domains with respect to each other inducing a slightly more closed overall complex. The observed differences between the behavior of CLD and the well studied related protein calmodulin are likely because of different physiological requirements for activation in vivo.