316 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
peptide, a random coil in solution, becomes helical in the presence of Ca 2+ -
CaM and attaches itself to the Ca 2+ - CaM N - and C - terminal hydrophobic
pockets through a large number of hydrophobic and some electrostatic
interactions.
The reference 85 authors found interproton distance restraints using
isotope - edited and isotope - fi ltered 2D, 3D, and 4D nuclear Overhauser effect
(NOE) experiments on a 1 : 1 complex of > 95%^15 N - and^13 C - labeled CaM and
the 26 - residue M13 peptide at natural isotopic abundance. The NOE experi-
ment is described in Sections 3.4.6 and 3.4.10. The one - letter amino acid
sequence of the M13 peptide is shown in Table 6.9. The 2D isotope - fi ltered
nuclear Overhauser enhancement spectroscopy (NOESY) experiments
yielded interproton distance restraints for the bound M13 peptide, suppressing
the signal of^13 C - and^15 N - attached protons. The^13 C - separated,^12 C - fi ltered 3D
NOESY spectra were used to identify NOEs between CaM protons and bound
peptide protons. Finally, 3D^13 C - and^15 N - separated and 4D^13 C –^13 C - separated
NOESY experiments were used to obtain NOEs between protein protons.^85
For details about the NMR methods, see Sections 3.4.6, 3.4.10, and 3.4.11. The
3D experiments showed intense NOE interactions between the M13 trypto-
phan (trp, W) residue at position 4 (1) and the methyl protons of CaM ’ s
met145. (See Table 6.9 for the numbering systems for the peptide positions.
The same experiment showed interactions between the aromatic ring protons
of phenylalanine (phe, F) at M13 ’ s position 8 (5) and the methyl group of
CaM ’ s ala88 and the C α H proton of ile85. These latter two CaM residues are
located near the C - terminal end of the central helix. The 4D experiments
yielded information on CaM ’ s shape in the complex. For instance, one result
showed that leu39 (N - terminal B helix) and val91 (C - terminal E helix) are
≤ 5 Å apart, indicating that the N - and C - terminal domains are in close proxim-
ity. This is compared to the Ca 2+ - CaM crystal structure where the methyl
groups of these residues are 30 – 33 Å apart. The authors state that the fl exibility
of the central loop allows the hydrophobic pockets of the two calmodulin
domains to approach each other so that the peptide ’ s hydrophobic residues
can form the many van der Waals contacts that hold the complex together
“ rather like two hands capturing a rope. ” Figure 2 of reference 85 illustrates
interpretations of the NMR data indicating at least 133 intermolecular interac-
tions between CaM and the M13 peptide. Several electrostatic interactions
between basic M13 arginine and lysine residues and CaM glutamatic acid
residues are also proposed by these authors. The most important M13 residues
for hydrophobic interaction with calmodulin appear to be those of trp4 (residue
1 the reference 87 numbering system) and phe17 (14), both of which make
many van der Waals contacts in the complex. Calmodulin and the M13 peptide
appear to be arranged in an antiparallel fashion — that is, trp4 (1) interacts with
residues (F89, F92, I100, L105, M109, V121, M124, I125, V136, F141, M144,
M145) in CaM ’ s C - terminal domain, whereas phe17 (14) interacts with resi-
dues (I27, L32, M36, M51, I52, V55, I63, F68, M71) in CaM ’ s N - terminal
domain. As will be seen below, similar arrangements are found for other pep-