Calmodulin-Peptide Binding

The cahnodulin-binding peptides assume random coil structures in solution, but in the presence of calmodulin they form amphipathic or amphiphilic (containing both polar and nonpolar residues) helices. All of these peptides have nanomolar (very high) affinities for calmodulin. Table 6.9 shows the primary amino acid sequence of some of the cahnoduUn-binding peptides, and it is informative to compare them as they are discussed in the following material. 

TABLE 6.9 Numbering System(s) for Calmodulin-Binding Peptides  

Argl6 (13) of the peptide chain forms electrostatic (hydrogen bonding and charge-coupling) interactions with the central loop bend and residues in 

Many more structure determinations of CaM-binding peptides have been carried out. For instance, the NMR structure determined by Ikura and co-workers for a CaM/CaMKK (from rat) complex (PDB ICKK) shows a calmodulin collapsed structure similar to those of CaM/smMLCK, CaM/ skMLCK, and CaM/CaMKIIa with the rCaMKK peptide cradled between calmodulin s C- and N-terminal domains. However, two features are different for CaM/rCaMKK. The first is that the peptide is bound in an inverted position compared to the others—that is, the N-terminal end of the rCaMKK peptide binds to CaM s N-terminal end and the C-terminal end binds to the C-terminal end rather than vice versa. This factor appears related to the clusters of basic residues on the target enzyme binding peptide—that is, when the 

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Peptide binding to calmodulin induces a large interdomain movement... 

Figure 6.21 Schematic diagram of the conformational changes of calmodulin upon peptide binding, (a) In the free form the calmodulin molecule is dumhhell-shaped comprising two domains (red and green), each having two EF hands with bound calcium (yellow), (b) In the form with bound peptides (blue) the a helix linker has been broken, the two ends of the molecule are close together and they form a compact globular complex. The internal structure of each domain is essentially unchanged. The hound peptide binds as an a helix.

A major limitation of the above studies of calmodulin-peptide interactions was that spectral evidence to support helix formation was limited to predictive algorithms and measurements of the difference in the circular dichroism of peptides and calmodulin in free solution and the CD in 1 1 complexes. Interpretation of such experiments was severely limited by the fact that calmodulin probably undergoes conformational changes upon binding peptides (Klevit et al., 1985). One elegant NMR study has been reported on a complex of melittin and bacterial-derived perdeuter-ated calmodulin the results were consistent with helix formation by the peptide in the complex (Seeholzer et al., 1986). 

The study of the interaction of peptides with proteins by optical spectroscopy is greatly facilitated if the peptide contains tryptophan and the protein does not, as in the case of the calmodulin/peptide systems studied here. Even for proteins which contain one or more tryptophans this approach can still be used provided that the spectral changes associated with binding of the peptide are sufficiently large. The use of synthetic peptides or site specific mutagenesis allows almost any residue to be replaced by tryptophan. The most conservative substitutions on the basis of size and hydrophobicity would be Phe -> Tip or Tyr -> Tip. 

Recently, a chameleon calcium reporter has been designed by R.Y. Tsien to sense calcium in cells by FRET. The structure of the system is based on a CEP separated from an YEP by the calmodulin calcium binding protein (CaM) and a calmodulin binding peptide (M13). If Ca ions are bound, CaM wraps around Ml 3 allowing high efficiency of excitation transfer from the donor CEP to the acceptor YEP (see Figure 9). The degree of FRET in chameleon is a sensitive ratiometric reporter (ratio 527/434 YPE/ CEP) of the calcium concentration in cells and in solution. The beauty of this approach is that the... 

Nonrepetitive but well-defined structures of this type form many important features of enzyme active sites. In some cases, a particular arrangement of coil structure providing a specific type of functional site recurs in several functionally related proteins. The peptide loop that binds iron-sulfur clusters in both ferredoxin and high potential iron protein is one example. Another is the central loop portion of the E—F hand structure that binds a calcium ion in several calcium-binding proteins, including calmodulin, carp parvalbumin, troponin C, and the intestinal calcium-binding protein. This loop, shown in Figure 6.26, connects two short a-helices. The calcium ion nestles into the pocket formed by this structure. 

Fig. 13. The binding sites of calcium in (a) parvalbumin (41a), (b) annexin (41) and (c) calmodulin (42). The drawings show two bidentate carboxylates coordinated to Ca2 in the EF-hand site of parvalbumin, and one bidentate carboxylate coordinated to Ca2 in annexin and calmodulin. All the donor atoms coordinated to the calciums are oxygen donor atoms from carboxylates of asp = aspartate, or glu = glutamate, or else peptide carbonyl oxygens from gly = glycine or met = methionine. Redrawn after Refs. (41-42).

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