Protoglobin: Structure and Ligand-Binding Properties - Advances in Microbial Physiology - page 84

structural re-arrangements that are indicated by the spectral changes
observed in the UV and Soret regions of

the CD spectra ( Ciaccio

et al., 2013 ).
At the quaternary structure level, Ma Pgb * is a dimer both in solution and
in the crystal ( Abbruzzetti et al., 2012; Nardini et al., 2008 ). The core of the
association interface ( > 2000 ˚ 2 ) is provided mostly by residues belonging to
the G- and H-helices, which build an inter-molecular four-helix bundle,
with the additional contribution of the Z-helices, and of the BC and FG
hinges ( Fig. 3.1 C). Overall, the quaternary structure of Ma Pgb * strongly
resembles that of the GCS globin domain ( Pesce et al., 2009; Zhang &
Phillips, 2003 ).
Molecular dynamics simulations revealed that the Ma Pgb * dimer is more
stable than the monomeric form, although the dissociation of the dimer into
separate monomers does not promote a substantial alteration in the overall
fold of the protein, but induces local re-arrangements in selected structural
elements, for instance, a relevant displacement of the G-helix and, to a lower
extent, of the H-helix. Such finding was not unexpected, as both helices are
directly involved in defining the dimeric four-helix bundle interface and dis-
sociation of the dimer increases dramatically their exposure to the aqueous
solvent, thus promoting conformational relaxation of the monomeric spe-
cies compared to the dimer. It is worth noting that the molecular dynamics
results indicate that access of ligands through the two tunnels is only granted
in the dimeric form, as tunnel 1 is primarily closed in the monomeric species.
Strikingly, however, when the same analysis is performed for the oxygen-
ated forms of the protein, significant structural re-arrangements are also
identified for the B- and E-helices. As both these helical segments delineate
the two tunnels in the Ma Pgb * protein matrix, it was suggested that ligand
migration is likely affected not only by the dimeric assembly of the protein
but also by the binding of ligands to the haem cavity ( Forti et al., 2011 ).

3. THE TWO-TUNNEL SYSTEM
Access of diatomic ligands, such as O 2 , CO and NO, to the fully bur-
ied Ma Pgb * haem is granted by two orthogonal apolar tunnels that reach the
haem distal region from entry sites at the B/G (tunnel 1) and B/E (tunnel 2)
helix interfaces ( Fig. 3.1 B). Notably, the topology of such two-tunnel sys-
tem is a unique feature of Pgb, totally unrelated with the ligand diffusion
path through the E7-gate first reported for Mb ( Bolognesi et al., 1982 ),