320 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
Small - angle X - ray scattering (SAXS) studies performed on complexes of CaM
with C20W and C24W indicated that the CaM – C24W complex had the same
globular shape as other CaM – CaM - binding peptide complexes discussed here
previously. However, the SAXS results for the CaM – C20W complex indicated
a more fl exible CaM molecule in solution, and its solution structure (described
next) illustrates the differences.
Griesinger, Krebs, and co - workers compared the structures of CaM – C20W
complex (PDB: 1CFF) with the CaM – skMLCK (also called M13) complex
(PDB: 2BBM) as well as with Ca 2+ - saturated calmodulin (PDB: 3CLN). Com-
parison of the M13 peptide sequence in Table 6.9 with that of C20W shows
that the second CaM - binding hydrophobic residue segment is missing in C20W.
This factor leads to signifi cant differences in the C20W – CaM three - dimen-
sional structure in comparison to that seen with the other peptides. Instead of
the globular structure noted for the CaM – skMLCK (M13), CaM – smMLCK,
and CaM – CaMKII complexes, the CaM – C20W structure resembles that of
Ca2+ - saturated CaM itself.
The C20W peptide, mainly random coil in solution, adopts an α - helical
structure when complexed, and it interacts with calmodulin in an antiparallel
manner — that is, the N - terminal half of the peptide binds to calmodulin ’ s C -
terminal domain and vice versa. However, the C20W peptide is missing the
second hydrophobic peptide (light shading in Table 6.9 ) and does not bind to
CaM ’ s N - terminal domain. In a 3D^12 C,^14 N ω 1 - fi ltered NOESY^1 H,^13 C HSQC
experiment, 49 intermolecular NOEs between peptide (unlabeled) and CaM
(^13 C - and^15 N - labeled) were defi ned. In a similar manner as observed with
other calmodulin - binding peptides, trp4 (1) showed numerous NOE interac-
tions to the methyl groups of ile100, leu105, met124, ile125, ala128, val136, and
met144 of CaM. In addition to trp4 (1), residues leu8 (5) and ile11 (8) on the
hydrophobic side of the peptide helix bind in calmodulin ’ s C - terminal hydro-
phobic pocket. Figure 8 of reference 87 illustrates the striking difference in
the number of peptide - CaM contacts when comparing the CaM – M13 complex
(that binds to both the C - and N - terminal domains of CaM) to CaM – C20W
(that binds only to the CaM C - terminal domain). All calmodulin C - terminal
met residues interact with the peptide, again because met side chains are fl ex-
ible, the C – S bond is long, and the sulfur atom is more polarizable than carbon,
leading to stronger van der Waals interactions. The central linker connecting
the two domains of calmodulin (in this case residues 74 – 84) is ill - defi ned and
does not appear in the structural representation.
Although previous research has indicated that target enzyme CaM - binding
domains must interact with both the C - and N - terminal domains of calmodulin
to end autoinhibition and activate the target enzyme, the reference 87 authors
have shown in their work that the Ca 2+ pump can be activated and autoinhibi-
tion by CaM - binding domains negated through binding to calmodulin ’ s C -
terminal domain only. Figure 11 of reference 87 graphically illustrates the
exposure of the CaM - binding domain ’ s tryptophan (trp4 (1)) residue to solvent
during the autoinhibition stage when the CaM - binding domain is attached to