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

Aalim M Weljie,Hans J Vogel

doi:10.1074/jbc.m311520200

Abstract

Calcium-dependent protein kinases (CDPKs) are an extensive class of multidomain Ca(2+)-regulated enzymes from plants and protozoa. In vivo the so-called calmodulin-like domain (CLD) of CDPK binds intramolecularly to the junction domain (JD), which exhibits both kinase-inhibitory and CLD binding properties. Here we report the high resolution solution structure of the calcium-regulatory region from soybean CDPK-alpha determined in the presence of a peptide encompassing the JD. The structure of both lobes of CLD resembles that of related helix-loop-helix Ca(2+)-binding proteins. NMR chemical shift mapping studies demonstrate that the JD induces significant structural changes in isolated Ca(2+)-CLD, particularly the C-terminal domain, although a stable complex is not formed. A CLD solution structure calculated on the basis of NMR data and long range fluorescence resonance energy transfer distances reveals an activated state with both lobes positioned side by side, similar to calcineurin B rather than calmodulin, highlighting the possible pitfall of assigning function purely from sequence information.

Highlights

Ca2ϩ signaling in plants underlies a necessity for a measured response to numerous biotic and abiotic stimuli such as drought, light, touch, and hormones [1,2,3]
NMR diffusion [16] and fluorescence resonance energy transfer (FRET) distance [17] data demonstrate that the calmodulin-like domain (CLD) shows significant compaction in the presence of Ca2ϩ as compared with the apoform, which is markedly different from CaM in which the two lobes remain independent in both the Ca2ϩ and apoforms
The activation properties of three soybean Calcium-dependent protein kinases (CDPKs) isoforms have been shown to be exquisitely sensitive to calcium concentration, and in turn calcium response has been shown to be sensitive to substrate [35]

Summary

Introduction

Ca2ϩ signaling in plants underlies a necessity for a measured response to numerous biotic and abiotic stimuli such as drought, light, touch, and hormones [1,2,3]. In the case of soybean CDPK-␣, bimolecular studies of a truncation mutant of the protein with the kinase domain, JD, and only the first two calcium-binding loops of the CLD cannot be activated intermolecularly by CaM, but significant activity is achieved with exogenous CLD [10]. 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. NMR diffusion [16] and fluorescence resonance energy transfer (FRET) distance [17] data demonstrate that the CLD shows significant compaction in the presence of Ca2ϩ as compared with the apoform, which is markedly different from CaM in which the two lobes remain independent in both the Ca2ϩ and apoforms

Methods

Results

Conclusion