Integration
Host Factor
Josh Weber '11 and Phan Truong '11
Contents:
I. Introduction
Integration host factor (IHF) was first discovered as a
host factor for bacteriophage λ integration. IHF was found
to assist in other processes that include high order protein-complexes
such as in replication where it binds to oriC, transcriptional
regulation where it can help initiate transcription by interacting with
RNA polymerase, and site-specific recombination (Goosen and van de
Putte, 1995). IHF’s primary function appears to be
architectural. It binds to DNA in a sequence specific manner
and introduces a sharp bend (>160°) in the DNA that
facilitates interaction between components in nucleoprotein array.
IHF is member of minor groove-intercalating
DNA-bending proteins. It is a small heterodimer with subunits that
are approximately 30% identical in sequence. The structure of IHF is
closely related to HU
, a nonspecific DNA binding protein. The members of the IHF/HU family
have been found in a broad range of prokaryotes (Oberto and
Rouviere-Yaniv, 1996).
II. General Structure
The fold of IHF
is
essentially the same as
that of HU
. The subunits of IHF, IHFα
and IHFβ,
are intertwined
to form a body with two
long β
sheet arms
that
extend from it
. The arms interact
exclusively with the minor groove of DNA and wrap around it. Each IHF
subunit contains 5 Beta-sheets(S)
and
3 Alpha-Helices(H)
.The
order of S and H is H1-H2-S1-S2-S'2-S'3-S3-H3
. The majority of the bending occurs at two kinks 9 base pairs(bp)
apart
and
proline residues at the tip of the arm intercalates between base pairs
.
The phosphate backbone contacts 26 positively
charged side chains
and
interacts with the N-termini
of all six
helices
of
the heterodimer. The ends of H1
and H3 form a clamp
by
binding to opposite sides of the minor groove with
respect to the intercalating proline.
H2 forms a hydrogen bond
from the bottom of the protein to adjacent DNA fragments.
III. Interaction of the
Beta-arms and Proline
The proline
residue in each arm is conserved
in every member of the HU/IHF families. Proline is favored because its
width allows extensive hydrophobic contact with the DNA bases
,
and its stubbiness allows formation of a hydrogen bond
between the peptide
backbone of the arm and N3
of A
immediately 5’ to each kink
.
It could also stabilize the structure of the turn at
the end of each
arm. The DNA within half a turn in either direction of the kinks adopts
a B-form structure with an unusually narrow minor groove.
IV. Clamping of
the DNA
The backbone on each side of the body of IHF
forms a tripartite clamp that binds across the minor groove. At the
center of this is a turn between S1
and S2
that lie
between two phosphates on opposite sides of the minor groove and is
hydrogen bonded to both strands by successive amide nitrogens
.
The
phosphates also interact with the N-termini of helices 1 and 3. On the
right side, the clamp straddles the second portion of the consensus
sequence and R46β extends from the S1-S2 turn, contacting
edges of this conserved sequence. On the left side, the clamp straddles
the minor groove of the A tract but doesn’t contact the
bases.
V. Recognition of
A-Tract
Regions
The A
tract is
straight, has a narrow minor
groove compared to a normal B-form minor groove
,
and high propeller twists between bases. It is
recognized by its structure, not by sequence, and is preferred by IHF
because the narrow minor groove fits into the clamp better. In this
tract and
the kinks,
is a spine of hydration, a
well-ordered string of water molecules. The
minor groove faces the protein and shields the spine from the solvent,
leaving unbounded hydrogen bond donors and acceptors. Side chain S47α,
from the S1-S2 turn, points into the minor
groove
and
provides hydrogen bond donors and acceptors
.
At
the top of the body, three β strands of
each subunit are
bridged by a row
of water molecules and the narrow minor groove lies parallel to these
strands. A thin sheet of water extends from the bridging subunits to
the DNA’s spine of hydration. The second consensus sequence
is in the exceptionally narrow minor groove and R46β replaces
the spine of hydration.
VI. Recognition
of Consensus Sequence
There are only two conserved sequences in the
IHF binding site
[concensus sequence]
. The first sequence is 5'-TATCAA-3'
and
is contacted by the arm of IHFα. R42β and
R46β reach into
the minor groove of the second concensus sequence, 5'-TTG-3',and
hydrogen
bond to the conserved bases. These contacts may help to select the
unusual DNA conformation that favors particular bases at these
positions. Base pairs 36 and 37 are the only bases that are conserved
in every binding site. These pairs are highly buckled because of the
intercalation of proline between bp 37 and 38. This buckling resolves
at bp 35 and creates a tilt of 32° between T35 and C36. The
conservation of A37 might be because of a need to satisfy a highly
distorted DNA structure and a limited number of protein contacts. The
large roll angle between bp 37 and 38 allows an interaction between
methyl groups in the minor groove: T37 is inserted into a hydrophobic
pocket between the ribose moiety and the methyl of T38. The arm of
IHFα is inserted into the minor groove more deeply than the
arm of IHFβ and to the lack of sequence specificity in this
region.
The second consensus sequence is recognized by
the body of IHF. R46β
extends from the S1-S2 turn into minor groove and
makes direct and indirect water mediated hydrogen bonds to the three
conserved bases. Its guanidinium is in the center of the groove and is
enforced by a chain of salt bridges. Both sides of the carboxylate of E44β
form salt bridges
,
one to the back of R46β
and
the other to R42β.
R42β
makes bridges to the
phosphate of A41.
E44β
acts as a buttress holding R46β
in place. The narrowness of the minor groove allows the
aliphatic portion of R46β
to make van der Waals ineractions
with ribose moieties of the DNA backbone on both sides of the grooves.
VII. Implication
IHF contributes to the biological functions
of many prokaryotes. One example is its role in the excision of λ prophage.
IHF bends the DNA, allowing the λ integrase to bind to its
promoter and contact the cleavage site. It has also been suggested that
IHF can assist RNA polymerase to initiate transcription by presenting a
portion of the promoter sequence to the relevant portion of RNA
polymerase. Since there is a large region of the major groove that is
left open for more interactions, other proteins may interact with these
sites. When IHF narrows the minor groove, it does so by widening the
major groove. When this occurs, these sites may adopt a better
conformation for other proteins to bind.
In order to bend DNA, proteins must overcome
the forces that usually keep the DNA straight. Some of the most
important of the forces that must be overcome are the repulsion of the
phosphate backbone charges and the energy from base stacking. By
placing a large positive surface on the inside of the DNA bend, IHF is
able to asymmetrically neutralize the double helix. To counteract the
energy of base stacking, IHF intercalates a hydrophobic side chain, the
proline at the tip of the β arms, between the base pairs.
The sequence specificity of the binding of IHF
to DNA is influenced by the sequence-dependent conformability of DNA,
shown by the amount of direct protein-DNA contacts made. Since the
minor groove has less unique features than the major groove, this theme
could be found in many minor groove binding proteins.
The conformation of the A tract that is
observed in the crystallization of the IHF-DNA complex is also observed
in the crystal structure of naked duplex DNA. The features of the A
tract are used to generate optimal contacts between IHF and the helix.
This provides evidence for the significance of structure of the A tract
as a recognition element for other proteins that interact with A-rich
sequences.
HU is 40% identical in sequence to IHF but
binds DNA nonspecifically, even though main features such as the
proline residue at the tip of each arm, and the distribution of
positive charge are conserved in each protein. The sequence specificity
of IHF must arise from the small differences between it and HU. In HU,
the R46 of IHFβ that contacts the conserved bases is replaced
with either valine or isoleucine. Structural differences in this region
also play a role in the sequence specificity of IHF. Helix 1 of
IHFα is displaced axially by 1.3 Å toward its
C-terminus, compared to HU and even IHFβ. This allows the
phosphate nearest its N-terminus to be tucked into the protein deeper,
resulting in an odd variation in twist at this position. The difference
in sequence specificity between the α and the β arms
of IHF could be explained by a difference in how the DNA is arranged in
the complex. The fact that the interactions between IHF and DNA are
asymmetric may drive the arms to making different interactions with the
minor groove. The α arm of IHF might be rigidified by unique
features in its amino acid sequence, such as P61 and P72, thereby
making it more effective in filtering DNA sequences.
VIII. Reference
Lemberg,
K., Rice, P., Swinger, K., and Zhang, Y. (2003). Flexible DNA bending in HU-DNA
cocrystal structures. The EMBO Journal 22:14,
3749-3760
Nash, H.A., Mizuuchi, K., Rice, P.A., and
Yang, S.-w. (1996). Crystal
structure of an IHF-DNA complex: a protein-induced U-turn.
Cell 87,
1295-1306
Goosen, N., and van de Putte, P. (1995). The regulation of
transcription initiation by integration host factor. Mol.
Microbiol.
16, 1-7
Oberto,
J., and Rouviere-Yaniv, J. (1996). Serratia
marcescens contains a
heterodimeric HU protein like Escherichia
coli and Salmonella tryphimurium.
J. Bact. 178, 293-297
Zullianello, L., van
Ulsen, P., van de Putte, P., and Goosen, N.
(1995). Participation
of the flank regions of the integration host
factor protein in the specificity and stability of DNA binding.
J.
Biol. Chem. 270, 17902-17907
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