title: Evidence for differing roles for each lobe of the calmodulin-like domain in a calcium-dependent protein kinase.
abstract: Calcium-dependent protein kinases (CDPKs) are structurally unique Ser/Thr kinases found in plants and certain protozoa. They are distinguished by a calmodulin-like regulatory apparatus (calmodulin-like domain (CaM-LD)) that is joined via a junction (J) region to the C-terminal end of the kinase catalytic domain. Like CaM, the CaM-LD is composed of two globular EF structural domains (N-lobe, C-lobe), each containing a pair of Ca(2+) binding sites. Spectroscopic analysis shows that the CaM-LD is comprised of helical elements, but the isolated CaM-LD does not form a conformationally homogeneous tertiary structure in the absence of Ca(2+). The addition of substoichiometric amounts of Ca(2+) is sufficient to stabilize the C-terminal lobe in a construct containing J and CaM-LD (JC) but not in the CaM-LD alone. Moreover, as J is titrated into Ca(2+)-saturated CaM-LD, interactions are stronger with the C-lobe than the N-lobe of the CaM-LD. Measurements of Ca(2+) affinity for JC reveal two cooperatively interacting high affinity binding sites (K(d)(,mean) = 5.6 nm at 20 mm KCl) in the C-lobe and two weaker sites in the N-lobe (K(d,mean) = 110 nm at 20 mm KCl). The corresponding Ca(2+) binding constants in the isolated CaM-LD are lower by more than 2 orders of magnitude, which indicates that the J region has an essential role in stabilizing the structure of the CDPK regulatory apparatus. The large differential affinity between the two domains together with previous studies on a plasmodium CDPK (Zhao, Y., Pokutta, S., Maurer, P., Lindt, M., Franklin, R. M., and Kappes, B. (1994) Biochemistry 33, 3714-3721) suggests a model whereby even at normally low cytosolic levels of Ca(2+), the C-lobe interacts with the junction, but the kinase remains in an autoinhibited state. Activation then occurs when Ca(2+) levels rise to fill the two weaker affinity binding sites in the N-lobe, thereby triggering a conformational change that leads to release of the autoinhibitory region.
authors.affiliation: Center for Structural Biology and Department of Biochemistry and Physics, Vanderbilt University, Nashville, Tennessee 37232-0146, USA.
authors.forename: John
authors.initials: J
authors.lastname: Christodoulou
authors.forename: Anders
authors.initials: A
authors.lastname: Malmendal
authors.forename: Jeffrey F
authors.initials: JF
authors.lastname: Harper
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Chazin
mesh_terms: D000595:Amino Acid Sequence; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000378:metabolism; D002614:Chelating Agents / Q000378:metabolism; D002942:Circular Dichroism; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012045:Regulatory Sequences, Nucleic Acid
chemical_list: D029681:Arabidopsis Proteins; D002118:Calcium; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002614:Chelating Agents; D020033:Protein Isoforms; D011494:Protein Kinases