Metalloproteins and Metallopeptides –Natural Metallofoldamers - Metallofoldamers: Supramolecular Architectures from Helicates to Biomimetics - page 7

network present in apo metallothionein which plays a role in the formation of a constant
but flexible backbone needed to adjust to the incoming metal to ensure specific metal
incorporation [30]. The metal-thiolate clusters then direct the “wrapping” of the protein
into its three-dimensional structure. There are two distinct metal binding sites in the C-
terminal (a-domain) and N-terminal domains (b-domain). The C-domain contains four
Zn 2þ ions bound to 11 Cys residues with five serving as bridging ligands, while the N-
domain binds three Zn 2þ ions via nine Cys residues and the metal binding follows a non-
cooperative model up to four equivalents, as shown by Co 2þ binding [31,32]. Computa-
tional studies also support this metal-binding mechanism. MM3/MD calculations of
metallothionein structure after sequential removal of metals show that the last two metals
bind to independent tetrathiolate sites from terminal thiolate ligands, which further sup-
ports independent Co 2þ binding to isolated sites prior to metal cluster formation and also
confirms that the C-terminal site is the first to bind metal ions [24]. Taken together, metal-
othionein represents an excellent example for describing the role of metal ions in the
proper folding of a natural metallofoldamer.
1.2.3 Conformational Change of Metalloproteins Caused by Ligand Binding
1.2.3.1 Calmodulin and Ca 2 þ / Ligand Binding
The role of metal ions in stabilizing the folded states of small proteins is well established,
as illustrated in zinc finger proteins [33] (Figure 1.1). Reversible binding of metal ions,
where both the metal-free disordered form and the metal-bound ordered form are func-
tional, is very widely observed among calcium-binding proteins. The coupling of the N-
and C-terminal lobes in the EF-hand Ca 2þ -binding calmodulin is a good example [34].
Since calcium signaling is such an important process in many metabolic systems, it is
likely that this kind of reversible order-disorder equilibrium is quite common. The binding
of up to four Ca 2þ ions in the two different globular ends of apo calmodulin [35] causes
significant conformational changes to the molecule (Figure 1.5), including straightening of
the long interdomain helix (Figure 1.5, red). Calcium binds to the sites with different affin-
ities (i.e., a higher preference for the C-terminal binding site than the N-terminus),
Figure 1.5 Right: structure of Ca 2þ -free calmodulin determined with NMR in solution, show-
ing a kink in the middle of the interdomain helix (PDB ID 1CFD). Left: crystal structure of
Ca 2þ -bound calmodulin, showing dramatic conformational change upon Ca 2þ binding
(PDB 1EXR).