326 GROUP I AND II METALS IN BIOLOGICAL SYSTEMS: GROUP II
is rotated about 80 ° compared to its fully calcium - saturated structure (PDB:
1CLL). Upon calmodulin binding, large EF movements in its large helical
domain (residues 660 – 800) and conformational changes in its segments desig-
nated as switches A, B, and C take place. At least 53 CaM residues interact
with 63 EF residues, leading to a large buried surface area of about 5900 Å^2.
This was the largest protein – protein interface between independently stable
CaM and EF molecules known at the time of this research. Figure 3 of refer-
ence 88 shows a representation of calmodulin binding to the anthrax exotoxin
and structural changes that occur upon CaM binding.
Several EF regions are important for describing the changes observed in
the EF protein in going from EF alone to the CaM - EF complex. Switch A (EF
residues 502 – 551) includes amino acid side chains that bind CaM. Switch B
(residues 578 – 591), disordered in the EF alone (PDB: 1K8T) structure, becomes
clearly visible — ordered — in the CaM – EF (PDB: 1K93) complex. Switch C
(residues 630 – 659) moves 33 Å from its EF alone position to its CaM – EF
complex position, and two switch C β - strands and connecting loop (642 – 652)
are converted to a helix in the CaM – EF structure. EF residues 501 – 540, com-
prising switch A and a region called C A , interact with central linker and C -
terminal CaM helices D, E, and F in a fashion similar to that described for
other systems described above with both hydrophobic and electrostatic inter-
actions. In particular, EF residue lys525 appears to be a “ hot - spot ” for CaM
binding as its mutation to alanine increases the concentration of CaM neces-
sary for EF activation 200 - fold for half - maximum response (EC 50 ). The
lys525ala mutant has little effect, however, on the enzyme - catalyzed reaction
at infi nite substrate concentration ( Vmax ). A second EF region, residues 615 –
634, 647 – 672 (EF helix L) and 695 – 721 (EF helix M), contacts three of the
four EF - hand motifs in CaM, one in the Ca 2+ - saturated C - terminal domain
and two in the calcium - free N - terminal domain. EF helices L and M contact
the fi rst EF - hand motif in calmodulin ’ s N - terminal domain and appear to sta-
bilize its conformation. The EF residue asp647 forms a salt bridge with calmod-
ulin ’ s arg90 stabilizing the interaction near EF - hand motif 3 in calmodulin ’ s
C - terminal domain. An asp647ala mutant has a signifi cantly lower V max but
shows little change in EC 50 in contrast to the lys525ala mutant. The authors
believe that the Michaelis – Menten behavior (see Section 2.2.4 ) indicates that
CaM binds residues in switch A before those in switch C — that is, binding
behavior is more important than catalytic competence for the switch A region
mutant and vice versa for the switch C mutation.
6.3.2.5 Conclusions. As Vetter and Leclerc state in reference 88 conclu-
sions: “ Target recognition and activation by CaM has been studied intensively
over the last two decades and today we are further away from a general model
than ever before. ” Much knowledge has been gained about calmodulin in the
work done to date; however, structures of calmodulin in complex with full -
length target enzymes, and not just with short peptides, are needed. The
structural studies on the calmodulin complex with the anthrax exotoxin are