| Ca2+-signalling is central to the reaction of plants to external stimuli. Calcium-dependent protein kinases (CDPKs) constitute a large protein family found only in plants and protozoa. Their response to calcium is mediated by a calcium-regulatory domain (CLD) comprised of four characteristic helix-loop-helix C2+-binding loops. CDPKs also have a kinase domain (KD), and a short junction domain (JD) which demonstrates both autoinhibitory and CLD-binding properties. The CLD and KD share significant sequence similarity to calmodulin (CaM) and CaM-dependent kinases (CaMKs), respectively, leading to the suggestion that the function of CDPK is mediated through a mechanism similar to CaM/CaMK. In this work, comparative modeling studies indicate that from a structural point of view this is a reasonable assumption for the JD/CLD interactions in soybean CDPK-α. However, subsequent NMR diffusion studies demonstrate that Ca2+-CLD is almost entirely collapsed in solution to form a nearly globular complex, in contrast to C2+-CaM which remains extended similar to its apo-form. Complete NMR structure determination of CLD in the presence of JD demonstrates that the structure of the CLD is globally similar to CaM, but several significant differences are observed. Firstly, the JD/CLD interaction is mediated by structural changes in the C-terminal domain. Second, the JD-CLD form does not form a stable complex, but demonstrates significant exchange properties. Finally, both the Ca2+- and JD-CLD forms show interactions between the two lobes of CLD, in a manner more analogous to calcineurin B than CaM. The collapse of both the Ca2+ - and JD-CLD was confirmed by fluorescence resonance energy transfer studies, where average distances between specific residues in the two lobes could be determined. The structure and backbone dynamics of the N-terminal domain of the CLD were also assessed in a sample in which the C-terminal domain was present, but not visible in the NMR spectra due to chemical exchange. This study confirms the presence of key residues which demonstrate significant exchange, and that the N-terminal “tether” region is highly mobile. Taken together, the results of this dissertation provide a basis for understanding the Ca2+-mediated activation of CDPKs, a possible target for rational herbicide and drug development. |
