Title and Abstract
Introduction
Assignment and Structure Determination
Comparison to other CaM/Peptide Complexes
Relaxation Time Measurements and Backbone Dynamics
Literature

Comparison of Structure to other CaM/peptide complexes

In contrast to the globular structure of CaM/M13 [2], the peptide C20W binds only to the C-terminal half of CaM (Figure 1). Only intermolecular NOE's between the C-terminal half of CaM and C20W were observed, but there is no evidence for any contacts between the N-terminal half and C20W (Figure 3 and 4). In addition, HN and Ca chemical shift differences between CaM/C20W and CaM are almost zero for the N-terminal domain, whereas both halves were affected when M13 and C21W are bound to CaM. This suggests a very different binding mode for C20W as compared to M13. Even though the major secondary structure elements of CaM do not change upon binding of either C20W or M13, the structures of CaM/C20W and CaM/M13 differ not only by their global structures, but there are also differences on a local level. The most significant local difference is observed for Trp 8 of the peptide. With the backbones of the C-terminal halves of the two complexes superimposed (rmsd=1.5), the plane of the indole ring in C20W is turned by almost 90^o with respect to the one in M13 (Figure 6). This difference in orientation also causes a large upfield shift (up to -1.3ppm) of the Indole ring protons in CaM/C20W (Figure 7).

Figure 6: Enlarged view of the tryptophan environments in CaM/C20W (a) and CaM/M13 (b). For comparable orientation the C-terminal backbones of CaM/C20W and CaM/M13 were superimposed. Experimental NOE's observed between protons of W4 in C20W and CaM are indicated as green lines in (a). The plane of the indole ring of W4 in CaM/C20W is rotated by almost 90^o with respect to the one in CaM/M13.

Figure 7: Secondary Chemical Shifts of the Tryptophane Protons of Calmodulin bound Peptides. Calculated Ring Current shifts are shown in Parantheses for CaM/M13 and CaM/C20W. Hz3 shows a significant upfield shift in CaM/C20W and TR2C/C20W.

This shift is consistent with aromatic ring current shifts induced by the adjacent Phe 92 residue of CaM. In the case of CaM/C20W the C-Hz3 bond is directed towards the center of the center of the aromatic ring in Phe92. Chemical shift calculations using the method of Case [5] within MolMol [6] also revealed Phe92 as the main contributor to the large upfield shifts. Similar shifts are found in a complex of TrC2, the C-terminal tryptic fragment of CaM, with C20W. The ability of CaM to bind a variety of peptides may arise from a fine adjustment of the methionine side chain conformations within CaM. A comparison of the conformations of the methionine residues in the binding pocket of the C-terminal domain (M109, M124, M144, M145) reveals large differences between CaM/C20W and CaM/M13. These differences result in large chemical shift d eviations between the two complexes (Figure 8). The eCH₃ methyl groups in the N-terminal half of CaM/C20W show chemical shifts comparable to free CaM, whereas the C-terminal domain exhibits a wide spread of chemical shifts. In CaM/M13, the e CH₃ shifts are well dispersed for both domains since both are involved in peptide bonding. However, due to the conformational differences in CaM/C20W and CaM/M13 the methionine eCH₃chemical shifts in the C-terminal half of these complexes are very different from each other.

Figure 8: Schematic ¹H,¹³C-HSQC spectra of the e CH₃ methyl groups of the Met residues. Arrows illustrate the chemical shift deviations for residues in CaM/C20W ( N-term., C-term) and CaM/M13 ( N-term.,C-term) with respect to CaM ( ).