Conformational Changes in the Ca2+-regulatory Region from Soybean Calcium-dependent Protein Kinase-α

Abstract

Calcium-dependent protein kinases are key proteins involved in plant and protozoal Ca 2؉ 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 Ca 2؉ -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 Ca 2؉ -saturated forms, as well as bound to a peptide encoding the JD sequence. Using an IAEDANS-modified Cys, a total of 15 Trp 3 Cys distances were measured in these three states from the five donor-acceptor combinations F334W-Cys 436 , L371W-Cys 436 , L403W-Cys 436 , F334W-L403C, and L371W-L403C. Consistent with recently reported NMR diffusion measurements and with 1 H, 15 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 2؉ , which is maintained on addition of the JD peptide. Interpretation of the distances suggests that the Ca 2؉ -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.Calcium-dependent protein kinases (CDPKs) 1 play a crucial role in the Ca 2ϩ -induced signaling pathways of both plants and protists (1-3). The CDPK response is calcium-dependent but calmodulin (CaM)-independent, presumably because of the presence of an internal Ca 2ϩ -regulatory region (calmodulinlike domain, or CLD) near the C terminus of CDPK. The CLD is very similar (Ͼ40% identity) to the ubiquitous calcium-regulatory protein CaM. The target for the CLD is another short domain found in CDPK entitled the junction domain (JD), which connects the CLD with the N-terminal protein kinase domain (4, 5).In vivo, increases in the cytosolic [Ca 2ϩ ] presumably are thought to induce a structural change directly from the apo state of the CLD to the activated (JD-CLD) form. Bimolecular studies (2, 6, 7) demonstrate that significant activity can be reconstituted using truncated CDPK constructs lacking the CLD with exogenous CLD in the presence of Ca 2ϩ . Although recent structural work demonstrates that the CLD shares the same global fold as CaM, it is clear that functionally (2, 6, 7) and structurally there are also some significant distinctions. 2 Like CaM (8 -10), CLD is comprised of two domains, each consisting of two helix-loop-helix EF-hand Ca 2ϩ binding loops (see Fi...

Unexpected Structure of the Ca2+-regulatory Region from Soybean Calcium-dependent Protein Kinase-α

“…The spatial orientation of the two lobes could not be precisely established based on NMR evidence alone; however, when combined with long range interdomain distances derived from FRET studies (17), a much clearer picture emerges. The open conformation uniquely was selected by the FRET distances, and hence we conclude that this is likely the significant population, acknowledging that the steady-state FRET restraints represent average distances.…”

“…This result suggests that the two domains of CLD are not independent and that the N-and C-lobes have an important role in binding. Furthermore NMR diffusion (16) and fluorescence resonance energy transfer (FRET) distance (17) data demonstrate that the CLD shows significant compaction in the presence of Ca 2ϩ as compared with the apoform, which is markedly different from CaM in which the two lobes remain independent in both the Ca 2ϩ and apoforms. Since structural and mechanistic understanding of CDPKs has thus far been limited to biochemical characterization, we were intrigued by the functional differences between the CLD and CaM and decided to examine the structural features of the calcium-regulatory region of soybean CDPK-␣.…”

“…The reader is referred to Ref. 17 for a more detailed derivation of the FRET-based distance restraints. FRET restraints were incorporated as target distances between the S/C ␥ side chain atoms for residues as reported (17) …”

Solution Structure and Backbone Dynamics of the N-Terminal Region of the Calcium Regulatory Domain from Soybean Calcium-Dependent Protein Kinase α

“…Diffusion analysis (13) and fluorescence resonance energy transfer studies (14) clearly indicate that the two lobes in the Ca 2+ -regulatory region of soybean CDPK-R are not independent in the Ca 2+ form. Hence having both the N-terminal lobe and C-terminal lobe attached is important and allows us to undertake a detailed examination of the N-terminal lobe while maintaining important global structural characteristics.…”

A New Assay Based on Fluorescence Resonance Energy Transfer to Determine the Binding Affinity of Bcl-xLInhibitors

“…Biochem., 72 (7), [1936][1937][1938][1939]2008 Note for Trp-AEDANS (22 Å ). [10][11][12] Moreover, the asparagine of Bak-BH3 peptide is not involved in the interaction with Bcl-x L . 5) Hence we replaced the asparagine with cysteine and labeled the fluorescence dye AEDANS at this residue.…”