Conformational changes in the Ca2+-regulatory region from soybean calcium-dependent protein kinase-alpha: fluorescence resonance energy transfer studies.
Calcium-dependent protein kinases are key proteins involved in plant and protozoal Ca2+ 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 N- and C-terminal lobes, each having two helix-loop-helix Ca2+-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 apo- and Ca2+-saturated forms, as well as bound to a peptide encoding the JD sequence. Using an IAEDANS-modified Cys, a total of 15 Trp --> Cys distances were measured in these three states from the five donor-acceptor combinations F334W-Cys436, L371W-Cys436, L403W-Cys436, F334W-L403C, and L371W-L403C. Consistent with recently reported NMR diffusion measurements and with 1H,15N heteronuclear correlation NMR spectra, the distances derived from fluorescence resonance energy transfer measurements in apoCLD indicate partial unfolding and a subsequent contraction on binding Ca2+, which is maintained on addition of the JD peptide. Interpretation of the distances suggests that the Ca2+-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 N- and 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.
Weljie
Aalim M
AM
Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Alberta T2N 1N4, Canada.
Robertson
Kindal M
KM
Vogel
Hans J
HJ
D020816:Amino Acid Motifs; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013025:Glycine max / Q000201:enzymology*
D002147:Calmodulin; D010455:Peptides; D017868:Cyclic AMP-Dependent Protein Kinases; D003545:Cysteine; D002118:Calcium