Introduction

Calmodulin (CaM) is an ubiquitous intracellular Ca²⁺ binding protein that contains two so called EF binding sites. It plays a pivotal role in Ca²⁺ regulation of a number of processes in eucaryotic systems. The crystal structure of CaM has been determined with high resolution revealing a dumbbell shape with two globular lobes at the N- and C- terminal side of the protein containing the Ca²⁺ binding domains (two in each) [1]. The two domains are connected with each other by a seven turn a helix which has shown to be highly flexible in solution (Figure 1a).

Figure 1:
a) Crystal structure of Ca2+-bound Calmodulin
b) Solution structure of CaM/C20W complex showing that C20W binds only to the C-terminal domain due to absence of N-terminal hydrophobic anchor residue.
c) Solution structure of CaM/M13 showing a tight globular complex involving C- and N-terminal domain. Extending the C20W sequence with ist N-terminal anchor should result in a binding mode of C21W similar to the one of M13.

CaM interacts with the amphipathic synthetic peptides such as M13 and CaMKII which resemble the calmodulin binding domain of myosin light chain kinase and calmodulin kinase II, respectively. These peptides have a high intrinsic tendency of helix formation but by themselves exhibit a random coil conformation in solution. Upon binding to calmodulin, however, they adopt a helical structure. The dumbbell conformation of CaM, on the other hand, collapses to a more globular conformation in which both domains of CaM both interact with the peptide [2](Figure 1c). SAXS studies of these complexes indicate the collapse of the calmodulin structure by a reduction of the radius of gyration from 62.5 Å down to 52.5 Å [3]. C20W is a peptide containing twenty amino acids which binds to CaM with high affinity (Kd = 11x10-9 mol l-1). It resembles the N-terminal residues of the CaM binding domain of the plasma membrane Ca2+ pump stopping just short before the anchor residue responsible for binding to the C-terminal half of CaM (Figure 2). C20W therefore interacts more strongly with the C-terminal domain of CaM (KD = 400x10^-9 mol^-1) as compared with the N-terminal domain (KD > 10^-6 mol^-1). This is consistent with the fact that the plasma membrane Ca2+ pump is activated by the C-terminal domain alone but not by the N-terminal domain. In contrast to the findings for all other complexes investigated thus far SAXS studies indicate that the CaM/C20W structure is not fully collapsed. A maximum length of 57.5 Å is found which is in between the values found for In this poster the solution structure of the complex CaM/C20W will be described.

Figure2: Sequence alignment of different calmodulin binding peptides
Hydrophobic anchor interacting with the C-terminal domain of CaM
Hydrophobic anchor interacting with the N-terminal domain of CaM