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10% sequence identity. However, major structural differences are present
between Ma Pgb * and the globin domain of GCSs at the 20 N-terminal loop,
which extend in opposite directions, and at the CE and FG loop regions
(where Ma Pgb * shows 6- and 12-residue insertions, respectively). These
differences have important structural implications for the haem-binding
pocket of the GCS globin domain, where the haem is solvent accessible
from the propionates edge, whereas specific mutations and local shifts of
the B-, E- and G-helices (particularly in G . sulfurreducens GCS, which is
hexacoordinated) prevent the formation of the apolar tunnels observed in
the Pgb fold.
The structural role of the Pgb-specific N-terminal region was analysed
by deletion mutants where (i) the first 20 residues are truncated
( Ma Pgb * - D N20 mutant) and (ii) the 20 N-terminal residues and the follow-
ing Z-helix, a protein stretch covering residues 1-33, are omitted ( Ma Pgb * -
D N20Z mutant) ( Ciaccio et al., 2013 ). A comparative analysis of the
Ma Pgb * - D N20 and Ma Pgb * crystal structures suggests that the
20 N-terminal residues are not required to attain the correct Pgb fold.
On the other hand, deletion of the 20 N-terminal residues partly uncovers
the haem cavity, which is instead blocked by such N-terminal segment in
the full-length protein. Such engineered haem accessibility may provide
an alternative route for haem/ligand exchange in Ma Pgb * - D N20, and,
indeed, it appears to facilitate haem access for an exogenous ligand, such
as azide, as well as the exchange of solvent molecules between the haem
pocket and the bulk solution ( Ciaccio et al., 2013 ). The structural details
provided by the crystallographic investigation are also in agreement with
CD spectra in the UV region, which suggest that the Ma Pgb * - D N20 mutant
maintains a secondary structure content matching that of Ma Pgb * . More-
over, the CD spectra in the Soret region indicate minor perturbation of
the haem cavity in Ma Pgb * - D N20, which could result from solvent expo-
sure or exchange in the haem region uncovered by deletion of
the
20 N-terminal residues.
On the contrary, in the Ma Pgb * - D N20Z mutant, a dramatic structural
change occurs upon removal of the 33 N-terminal residues, such that the
protein appears to collapse, partly bringing about a relevant alteration of
the haem distal side, with the likely formation of a hexacoordinated haem,
both in the ferric and ferrous species, and direct implications for ligand bind-
ing. Indeed, modelling studies indicate that the contemporary absence of the
N-terminal loop and of the Z-helix would expose to the solvent a wide
hydrophobic surface from the protein core, thus inducing the dramatic
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