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topological positions B9, B10 and E11 that appear to be involved not only
in ligand recognition and binding but also in the dynamic regulation of
ligand exchange between the solvent and the distal pocket.
Phe(93)E11 appears to play a role in ligand sensing and discrimination.
In fact, the Phe(93)E11 side chain rotates by
120
, relative to ligand-free
ferric
Ma
Pgb, upon cyanide binding (
Fig. 3.2
B); a similar side chain rota-
tion occurs upon azide and imidazole binding, but it is somehow impaired
in the case of nicotinamide. This ligand-linked conformational change
makes room for a consequent “induced” conformational re-arrangement
of Trp(60)B9 side chain, which inserts into the distal site and hydrogen
bonds the Fe-bound ligand. Such a sequential mechanism is confirmed
by the structure of the ferric cyanide-bound Trp(60)B9Ala mutant, where
the ligand is coordinated to the haem-Fe atom and stabilised only by the
Tyr(61)B10 OH group, the Phe(93)E11 residue being rotated by about
120
relative to ligand-free
Ma
Pgb
*
, even in the absence of Trp(60)B9.
The structure of ferric
Ma
Pgb
*
in complex with nicotinamide reveals that
despite the 60
rotation of Phe(93)E11 side chain, Trp(60)B9 does not
enter the haem distal cavity, thus indicating that there must be a certain
rotation of Phe(93)E11 ligand sensor to trigger the conformational change
of Trp(60)B9.
Sensitivity response of Phe(93)E11 to the haem-bound ligand is consis-
tently observed also through molecular dynamics simulations, which
evidenced how the haem-bound ligand introduces steric hindrance onto
the side chain of Phe(93)E11 (
Forti et al., 2011
). However, the ligand-
sensing role of Phe(93)E11 does not necessarily require the presence of
an aromatic residue at the E11 topological position (although Phe is con-
served in Pgbs), as demonstrated by the structure of the cyanide-bound
Phe(93)E11Leu mutant, where the ligand-binding mode and the distal site
geometry are essentially identical to those found in the
Ma
Pgb
*
-cyanide
structure. Cyanide dissociation rate constants, measured in solution for ferric
Ma
Pgb
*
and selected mutants, are in good agreement with the ligand-
stabilisation events and mechanisms described by the crystal structures
(
Pesce, Tilleman, et al., 2013
).
A relevant finding (probably the most important) is that the conforma-
tional adaptability shown by
Ma
Pgb
*
haem distal site residues results in
coupling between ligand sensing/binding and haem distal site accessibility
through the two-tunnel system, as Trp(60)B9 and Tyr(61)B10 side chains
line the two separate tunnels. Thus, the comparison of ferric
Ma
Pgb
*
in
its liganded and unliganded states identifies two distinct haem distal site
