Comparative modeling studies of the calmodulin-like domain of calcium-dependent protein kinase from soybean.

Calmodulin-like domain protein kinases (CDPKs) represent a new class of calcium-dependent protein-phosphorylating enzymes that are not activated by calmodulin or phospholipid compounds. They have been found exclusively in plant and protozoal tissues. CDPKs are typified by four distinct domains: an N-terminal leader sequence, a protein kinase (PK) domain, a calmodulin-like domain (CLD), and a junction domain (JD) between the PK domain and CLD. Structural characterization of the CLD of CDPKalpha from soybean was undertaken based on the amino acid sequence homology of CLD to the structurally well-characterized calmodulin (CaM) family of structures. Tertiary models of apo-CLD, Ca(2+)-CLD complex, and intermolecularly bound Ca(2+)-CLD-JD complexes were obtained via automated and non-automated homology building methods. The resulting structures were compared and validated based on energy differences, phi-psi angle distribution, solvent accessibility, and hydrophobic potential. Circular dichroism, one-dimensional, and two-dimensional nuclear magnetic resonance spectroscopy studies of the CLD and peptides encompassing the JD provide experimental support to the models. The results suggest that there is a possible interaction between the CLD and JD domain similar to that of the CaM/calmodulin-dependent protein kinase II system. At low Ca(2+) levels, the JD may act as an autoinhibitory domain for kinase activity, and during calcium activation an intramolecular CLD-JD complex may form, relieving inhibition of the PK domain. Interactions between the JD and the C terminus of the CLD appear to be particularly important. The outcome of this study supports an intramolecular binding model for calcium activation of CDPK, although not exclusively.

Tryptophan fluorescence of calmodulin binding domain peptides interacting with calmodulin containing unnatural methionine analogues.

The interactions between the abundant methionine residues of the calcium regulatory protein calmodulin (CaM) and several of its binding targets were probed using fluorescence spectroscopy. Tryptophan steady-state fluorescence from peptides encompassing the CaM-binding domains of the target proteins myosin light chain kinase (MLCK), cyclic nucleotide phosphodiesterase (PDE) and caldesmon site A and B (CaD A, CaD B), and the model peptide melittin showed Ca(2+)-dependent blue-shifts in their maximum emission wavelength when complexed with wild-type CaM. Blue-shifts were also observed for complexes in which the CaM methionine residues were replaced by selenomethionine, norleucine and ethionine, and when a quadruple methionine to leucine C-terminal mutant of CaM was studied. Quenching of the tryptophan fluorescence intensity was observed with selenomethionine, but not with norleucine or ethionine substituted protein. Fluorescence quenching studies with added potassium iodide (KI) demonstrate that the non-native proteins limit the solvent accessibility of the Trp in the MLCK peptide to levels close to that of the wild-type CaM-MLCK interaction. Our results show that the methionine residues from CaM are highly sensitive to the target peptide in question, confirming the importance of their role in binding interactions. In addition, we provide evidence that the nature of binding in the CaM-CaD B complex is unique compared with the other complexes studied, as the Trp residue of this peptide remains partially solvent exposed upon binding to CaM.

Tryptophan fluorescence quenching by methionine and selenomethionine residues of calmodulin: orientation of peptide and protein binding.

The two interaction surfaces of the dumbbell-shaped calcium-regulatory protein calmodulin (CaM) are rich in the amino acid Met. In this work we have used fluorescence spectroscopy to study the role of these Met residues in binding the single Trp residue that is found in many CaM-binding domain peptides. This approach is facilitated by the absence of Trp residues in CaM. In addition to the wild-type protein, we studied CaM containing the unnatural amino acid selenomethionine (SeMet), which was biosynthetically substituted for its nine Met residues. Furthermore, a CaM mutant protein in which all four Met residues in the C-terminal domain were mutated to Leu, and the N-terminal domain contained either Met or the unnatural SeMet, was studied. The Trp fluorescence quantum yield of many Trp-containing CaM-binding peptides increases upon binding to calcium-CaM. Moreover, the emission wavelength of the Trp fluorescence is blue-shifted from 353 to 325-333 nm. These parameters indicate movement of Trp from a solvent exposed to a hydrophobic environment. The fluorescence results obtained with these four CaM variants showed that Se is very effective at quenching Trp fluorescence in the calmodulin-bound peptides from myosin light chain kinase (MLCK) and CaM kinase I, while S is somewhat effective (Se > S > C). The quenching effect is markedly distance dependent, as it only influences the Trp residue of the bound peptide (<=7 A) but has little effect on the two Tyr residues in the C-terminal domain of CaM (>=10 A). Since the Trp fluorescence quenching is very dramatic, the protein containing Leu's in the C-terminal domain and SeMet's in the N-terminal domain allowed us to directly determine the orientation of the MLCK and CaM kinase I peptides bound to CaM; in both cases the Trp residue binds to the C-terminal domain of CaM. Our data indicate that SeMet quenching of Trp fluorescence could become a simple and useful tool for studies of protein folding, and protein-protein and protein-peptide interactions.