Biology Reference
In-Depth Information
The CooA F-helix is responsible for the direct DNA binding. On the
helix-turn-helix motif (EF-region), three residues have been identified as
responsible for the site-specific recognition of DNA: Arg177, Gln178,
and Ser181. Their side chains form hydrogen bonds with specific DNA pairs
on the CooA-dependent promoter binding site (
Aono et al., 1999
).
With spectroscopic and mutagenesis techniques, His77 has been identi-
fied as critical for conformational changes upon CO binding. These struc-
tural changes occur at the haem-binding domain level and are subsequently
communicated to the DNA-binding domain, thus allowing the sequence-
specific interaction with DNA (
Shelver et al., 1999; Uchida et al., 2000;
Vogel, Spiro, Shelver, Thorsteinsson, & Roberts, 1999
).
The crystal structure of the reduced (Fe
2
þ
)
Rr
CooA shows a homo-
dimeric form in which the haem-Fe axial ligands are the proximal His77
of the same subunit and the distal Pro2 N-terminal nitrogen from the oppo-
site partner subunit (
Fig. 1.3
A) (
Lanzilotta et al., 2000
). Confirming spectro-
scopic and mutation results, NMR experiments on His77 and Pro2 mutants
highlight the binding of CO at the haem distal site in correspondence of
Pro2. Therefore, the suggested role for Pro2 is to direct the ligand towards
the appropriate binding site (
Clark et al., 2004; Yamamoto et al., 2001
).
The arrangement of the homo-dimer in the crystal displays two symmet-
ric haem-binding domains coupled via their C-helices to two asymmetric
DNA-binding domains (
Fig. 1.3
B). A comparison with the more symmetric
CRP structure suggests that the binding of CO to the haem would cause
rearrangements in the C-helix and, consequently, a more symmetric
arrangement of the DNA-binding domain (
Akiyama, Fujisawa, Ishimori,
Morishima, & Aono, 2004; Kerby, Youn, Thorsteinsson, & Roberts,
2003; Lanzilotta et al., 2000
). In agreement with this, mutations of residues
at the dimer interface, and in particular, the mutation of Gly117 in the
C-helix into bulky residues, disrupt the structural modifications that happen
upon CO binding, thus, creating inactive CooA (
Youn et al., 2001
). When
CO binds to the haem, structural rearrangements cause a roll-and-slide
mechanism in which the C-helix moves towards the haem, thus, forming
a wide hydrophobic pocket around the ligand (
Yamashita, Hoashi,
Tomisugi, Ishikawa, & Uno, 2004
).
Kinetics measurements on the
Rr
CooA homo-dimer show positive
cooperativity for CO binding (Hill coefficient
n
¼
1.4), low CO affinity
1.3
m
M
1
), and slow conformational
transitions between an open and a closed form that convert the DNA-
binding domains from the inactive to the active state. All these features
0.16
m
M
1
,
K
2
(
P
50
¼
2.2
m
M,
K
1
¼
¼
