Báo cáo khoa học: Structure of FocB – a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli pdf

Structure of FocB – a member of a family of transcription
factors regulating fimbrial adhesin expression in
uropathogenic Escherichia coli
Ulrika W. Hultdin
1
, Stina Lindberg
2
, Christin Grundstro
¨
m
1
, Shenghua Huang
1
, Bernt Eric Uhlin
2
and A. Elisabeth Sauer-Eriksson
1
1 Department of Chemistry, Umea
˚
University, Sweden
2 Department of Molecular Biology, The Laboratory for Molecular Infection Medicine Sweden (MIMS), Umea
˚
University, Sweden
Introduction
Fimbriae, which are long, adhesive, polymeric protein
structures, are expressed on the surface of many patho-
genic bacteria. Uropathogenic Escherichia coli (UPEC),
the primary cause of urinary tract infections, express
various types of fimbriae for adhering to (and some-
times invading) host cells [1,2]. Because the interaction
between the fimbrial adhesins and their receptors is

specific, the expression of different types of fimbriae
makes infection progress in different niches of the
urinary tract.
Keywords
fimbriae; FocB; repressor protein;
uropathogenic Escherichia coli; X-ray
crystallography
Correspondence
A. E. Sauer-Eriksson or B. E. Uhlin,
Department of Chemistry, Umea
˚
University,
SE-90187 Umea
˚
, Sweden; Department of
Molecular Biology, Laboratory for Molecular
Infection Medicine Sweden (MIMS), Umea
˚
University, SE-901 87 Umea
˚
, Sweden
Fax: +4690 7865944; +4690 772630
Tel: +4690 7865923; +4690 7856731
E-mail: elisabeth.sauer-eriksson@
chem.umu.se;

Database
The atomic coordinates and structure
factors for the Escherichia coli FocB
protein are available in the Protein Data

Bank database under the accession number
3M8J
(Received 28 January 2010, revised 5 May
2010, accepted 17 June 2010)
doi:10.1111/j.1742-4658.2010.07742.x
In uropathogenic Escherichia coli, UPEC, different types of fimbriae are
expressed to mediate interactions with host tissue. FocB belongs to the
PapB family of transcription factors involved in the regulation of fimbriae
gene clusters. Recent findings suggest that members from this family of
proteins may form homomeric or heteromeric complexes and exert both
positive and negative effects on the transcription of fimbriae genes. To
elucidate the detailed function of FocB, we have determined its crystal
structure at 1.4 A
˚
resolution. FocB is an all a-helical protein with a helix-
turn-helix motif. Interestingly, conserved residues important for
DNA-binding are located not in the postulated recognition helix of the
motif, but in the preceding helix. Results from protein–DNA-binding stud-
ies suggest that FocB interacts with the minor groove of its cognate DNA
target, which is indicative of a DNA interaction that is unusual for this
motif. FocB crystallizes in the form of dimers. Packing interactions in the
crystals give two plausible dimerization interfaces. Conserved residues,
known to be important for protein oligomerization, are present at both
interfaces, suggesting that both sites could play a role in a functional FocB
protein.
Structured digital abstract
l
MINT-7901626: focB (uniprotkb:Q93K76) and focB (uniprotkb:Q93K76) bind (MI:0407)by
x-ray crystallography (
MI:0114)

Abbreviations
CRP, cAMP receptor protein; HNF, hepatocyte nuclear factor; HTH, helix-turn-helix; PDB, Protein Data Bank; UPEC, uropathogenic
Escherichia coli.
3368 FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS
To balance metabolic efficiency, the expression of
the different fimbrial types needs to be regulated. The
control mechanisms are complex and act at multiple
levels. Environmental factors may affect the on–off
switching of the fimbrial genes indirectly by influencing
the expression patterns of certain proteins. In addition,
there is cross-regulation between the different fimbrial
gene clusters [3–5].
Many types of fimbriae expressed by UPEC strains
are structurally similar and their genetic organizations
show high resemblance. The P fimbriae, commonly
associated with pyelonephritis [6], are the most studied
and have been described extensively. The pap gene
cluster, sufficient for the production of P fimbriae,
consists of nine structural and two regulatory genes.
The structural genes for P fimbriae encode both minor
and major subunits, building up the fimbrial rod, as
well as proteins important for the transport and assem-
bly of the fimbriae at the bacterial surface (Fig. 1).
Recent studies have revealed additional genes in the
promoter distal region of the main fimbrial operons,
and their role also appears to be at the regulatory level
[7,8].
The pap genes are transcribed from two divergent
promoters, P
B

and P
I
, separated by an intercistronic
region containing binding sites for the PapB protein,
which is a key factor in the regulation of fimbriae
[9,10]. Other important regulators that operate in this
region include cAMP receptor protein (CRP), Lrp,
PapI, DNA adenine methylase and histone-like nucle-
oid-structuring protein [11].
UPEC commonly express F1C fimbriae, a fimbrial
type often found in strains also carrying genes for type 1
or P-fimbrial expression [12]. The receptors for F1C
fimbriae have been identified as glycosphingolipids
[13,14] and their specific interaction allows the F1C
fimbriae to adhere to the collecting ducts and distal
tubules of the kidney [15].
The organization of the genetic determinant for F1C
fimbriae, the foc operon, is very similar to that of the
pap operon, with only a few transposed genes. The foc
gene cluster includes seven structural and two pro-
pap
J96
Usher AdhesinChaperone Minor subunits
I
B
A
E
GF
K
D

J
H
C
Y X
Regulation (?)
Regulators
Major
prs
J96
I B
A
E GFKD
J
H
C
X
subunit
prf
536
I B
A
E GFKD
J
H C
X
sfa
IHE3034
C B A S HGED F Y X
foc
536

I
B A
F
HG
C
I D
Y
X
HNS
CRP
Lrp
H-NS
focB
focI
P
B
P
I
+
–
Site 2Site 1
B
+
–
Fig. 1. Genetic organization of different fimbrial gene clusters: pap (P fimbriae), prs (Prs fimbriae), prf (P-related fimbriae), sfa (S fimbriae)
and foc (F1C fimbriae). A representative E. coli strain carrying the gene cluster is indicated. The transcription factor FocB is acting in the int-
ercistronic region between the focI and focB genes, together with a number of other regulatory proteins. FocB has also been described to
have a repressive effect on fim expression (encoding type 1 fimbriae) [5]. Boxes with the same color represent genes with similar function.
Recent findings suggest that fimbrial gene clusters also include the previously unrecognized novel regulatory genes, X and Y [8]. Modified
from Sjo

¨
stro
¨
m et al. [8].
U. W. Hultdin et al. Structure of FocB
FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS 3369
moter-proximal regulatory genes [16,17] (Fig. 1). One
of these regulatory genes encodes the FocB protein, a
member of the PapB family of fimbrial transcriptional
factors.
The members of the PapB family identified so far
exist in both the E. coli and Salmonella species and are
all involved in the regulation of fimbrial expression. At
the amino acid level, FocB is 81% identical to PapB
and 100% identical to SfaB, the protein regulating the
expression of S-fimbriae [17], both of which are associ-
ated with E. coli newborn meningitis strains [18]. FocB
is also 47% identical to the PefB protein of Salmo-
nella typhimurium [19], and 34% and 28% identical to
the FaeB and FanB proteins, respectively. The latter
two proteins are regulators of the K88 and K99 fimbri-
al types that are expressed by enterotoxigenic E. coli
and cause diarrhea in domestic animals. In many
respects, the roles of the FaeB and FanB proteins in
transcriptional regulation are similar to that of PapB
in the regulation of the pap operon [20,21].
UPEC strains often carry several different fimbrial
operons within their genome [12]. For example, the
UPEC strain J96 carries at least the operons fim, pap,
prs and foc [22–24]. However, less than 10% of the

bacteria display more than one fimbrial type on their
surfaces simultaneously. Thus, fimbrial expression
involves cross-regulatory interactions between the dif-
ferent operons. Recent experiments suggest that an
intricate hierarchy exists with respect to the the cross-
regulation of the pap and foc operons because FocB
could stimulate the expression of pap, whereas PapB is
insufficient for stimulating the expression of foc by
itself [5]. In concordance, the PapB family members
share a common core structure, as revealed by multiple
sequence alignments. These homologous proteins share
almost completely conserved regions that are known to
be important for oligomerization and DNA-binding
[25].
In the present study, we describe the structure of
FocB, which is the first reported structure of a member
of the PapB protein family. FocB crystallizes as
dimers, in which each subunit contains one helix-turn-
helix (HTH) motif. Crystal packing interactions
suggest that there are two dimerization interfaces of
interest for the in vivo function of FocB. Amino acids
known to be important for DNA-binding are exposed
on the protein surface, although they are not located
in the recognition helix of the HTH motif. The results
obtained from DNA-shift assays suggest that FocB
binds to the DNA minor groove, thus indicating a
DNA-binding pattern different from that of classical
HTH motifs.
Results
Structure determination

The E. coli FocB structure (109 amino acid residues)
was determined at a resolution of 1.4 A
˚
by multiwave-
length anomalous diffraction [26] from a single crystal
of the selenomethionine labeled protein. The crystal
comprised two molecules per asymmetric unit. Apart
from several residues at the N- and C-terminal ends,
all protein residues could be modeled into the electron
density. The final model contains residues 10–99 of
chain A and residues 10–97 of chain B. The final
R-values, R
work
= 0.196 and R
free
= 0.222, are higher
than expected for this resolution. This is probably a
result of the additional electron density ascribed to the
N- and C-terminal residues of the protein, which were
not modeled in the structure because of disorder.
Table 1 summarizes the statistics of X-ray data collec-
tion and the results for the structural refinement of
FocB. The coordinates and structure factors are depos-
ited in the Protein Data Bank (PDB) (accession code
3M8J).
The structure of FocB
As anticipated from CD measurements and secondary
structure prediction software [27] (Fig. 2), the amino
acid chain of FocB forms an all a-helical structure
that comprises five a-helices: a1 (Asp12-Ser21),

a2 (30Glu-Ser40), a3 (Asp45-Gly58), a4 (Arg61-Tyr68),
and a5 (Asn71-Tyr95) (Fig. 3). Of these, helices a4 and
a5 and the connecting turn (Gln69-Asn71) comprise an
HTH motif, which appears to be quite different from
the canonical HTH motifs found in many bacterial
transcription factors [30–32]. In particular, the second
helix of the HTH motif, the putative recognition helix,
is much longer in the FocB structure than in a typical
HTH motif. Structural similarities with the helix-
loop-helix motif in homeodomains also strengthen the
view that FocB harbors an atypical HTH motif.
The helix a5, at the center of the domain, can be
divided into two parts based on its amino acid compo-
sition. The N-terminal part, comprising residues Asn72
to Asn86, is amphiphatic, whereas the C-terminal part,
starting at residue Val87 and extending to the last mod-
eled residue Tyr95, is predominantly hydrophobic in
nature, with the exception of one residue, Arg91. The
hydrophobic surface of the amphiphatic N-terminal
part of the helix is shielded from the solvent by three
short anti-parallel helices a2–a4 situated perpendicular
to a5. The interactions between these four helices form
Structure of FocB U. W. Hultdin et al.
3370 FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS
Table 1. Data collection and refinement statistics.
Data collection Native Peak Inflection Remote
Wavelength (A
˚
) 0.9792 0.9792 0.9795 0.9754
Unit cell P2

1
2
1
2
1
P2
1
2
1
2
1
P2
1
2
1
2
1
P2
1
2
1
2
1
Unit cell parameters (A
˚
) a = 47.39 a = 47.8 a = 47.8 a = 47.8
b = 58.82 b = 59.3 b = 59.3 b = 59.3
c = 65.84 c = 66.1 c = 66.2 c = 66.2
Range of resolution (A
˚

) 47.40–1.40 ⁄ (1.47–1.40) 47.77–1.90 ⁄ (2.00–1.90) 47.80–2.00 ⁄ (2.11–2.00) 47.77–2.05 ⁄ (2.16–2.05)
R
merge
a
0.036 ⁄ (0.314) 0.058 ⁄ (0.381) 0.055 ⁄ 0.335) 0.056 ⁄ (0.428)
Number of observations 230628 213993 187663 174385
Number of unique reflections 36163 15361 13252 12331
completeness (%) 97.8 ⁄ (97.4) 100.0 ⁄ (100.0) 100.0 ⁄ (100.0) 99.9 ⁄ (100.0)
I ⁄ rI 28.6 ⁄ (6.1) 29.7 ⁄ (6.6) 32.6 ⁄ (8.3) 33.0 ⁄ (6.7)
Redundancy 6.4 ⁄ (6.4) 13.9 ⁄ (12.5) 14.2 ⁄ (14.5) 14.1 ⁄ (14.4)
Anomalous completeness (%) 99.9 ⁄ (99.9) 99.9 ⁄ (100.0) 99.9 ⁄ (100.0)
Anomalous multiplicity 7.5 ⁄ (6.5) 7.6 ⁄ (7.6) 7.6 ⁄ (7.6)
Refinement
Number of reflections 34198
R-factor
b
0.196
R
free
c
0.222
Number of atoms 1539
Number of water molecules 148
Overall mean B (A
˚
2
) 17.3
Protein atoms (A
˚
2

) 15.8
Water molecules (A
˚
2
) 28.1
rmsd bond length (A
˚
) 0.017
rmsd bond angles (°) 1.61
Ramachandran plot favoured (%) 100
Ramachandran plot accepted (%) 0
Ramachandran plot outliers (%) 0
PDB code 3M8J
a
For replicate reflections, R = RI
hi
) <I
h
>| ⁄ R<I
h
>; I
hi
= intensity measured for reflection h in data set i,<I
h
> = average intensity for reflection
h calculated from replicate data.
b
R-factor = R||F
o
| ) |F

c
|| ⁄ R|F
o
|; F
o
and F
c
are the observed and calculated structure factors, respectively.
c
R
free
is based upon 5% of the data randomly culled and not used in the refinement.
Fig. 2. Sequence alignment (BLAST) [28] of FocB and PapB from E. coli. The sequence identity is 81%. Residues not identical are highlighted
in yellow. Secondary structural elements from the current structure of FocB are shown in bold with the HTH motif highlighted in red. The
first and last residue visible in the electron density of the FocB structure is highlighted in blue. Residues Arg61 and Cys65, which were previ-
ously found to be important for DNA-binding, are boxed in pink, whereas some residues previously found to be important for oligomerization
are boxed in green [25]. The secondary structure elements predicted with
JPRED3 [29] are shown in blue (H, helices; E, b-strands). Interest-
ingly, the a1 helix in FocB was predicted to be a b-strand in PapB.
U. W. Hultdin et al. Structure of FocB
FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS 3371
a stable hydrophobic core. The first helix a1 is posi-
tioned perpendicular to a5, but situated on the oppo-
site side of helix a5 with respect to helices a2–a4. Only
a few contacts are formed between a1 and a5, and
include one hydrogen bond between the main chain
nitrogen atom of Leu23 and the side chain Oc1 atom
of Thr83, and one hydrophobic interaction between
the side chain of Leu18 and the Cc2 atom of Thr83.
FocB dimerization

Cross-linking and size exclusion chromatography stud-
ies showed that FocB forms predominantly dimers in
solution [27]. Furthermore, the packing of molecules in
the crystal structure suggests that FocB is dimeric. The
asymmetric unit comprises two molecules of FocB.
Crystal packing contacts provide two alternative possi-
bilities for homodimeric FocB interactions. At the first
dimer interface, the two monomers, chains A and B in
the asymmetric unit, form extensive contacts between
their helices a2 and a5(Fig. 4A, subunits colored in
light and dark blue). These helices are positioned per-
pendicular with respect to each other in a four-helix
arrangement. The two monomers are related by a non-
crystallographic two-fold symmetry operation, and we
refer to this interface as dimer Interface-I. The inter-
Fig. 3. Ribbon representation of the structure of FocB. Helices
a1–a5 are displayed, with the HTH motif highlighted in blue. The
conserved DNA-binding residues Arg61 and Cys65 are shown as
ball and sticks.
A
BC
Fig. 4. Crystal packing of the FocB struc-
ture. (A) Subunits forming Interface-I are
colored in light and dark blue; subunits of
Interface-II are colored in light and dark
coral. (B) Interactions in Interface-I.
(C) Interactions in Interface-II. For clarity,
only selected residues are shown as sticks.
Selected hydrogen bonds are shown in
green (dotted line).

Structure of FocB U. W. Hultdin et al.
3372 FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS
face involves predominantly residues positioned at the
hydrophobic C-terminal part of a5. An extensive
hydrophobic core is formed over this interface, includ-
ing four residues from a5 (AB-Leu85, AB-Leu88,
AB-Val89 and AB-Leu92) and two residues from a2
(AB-Leu35 and AB-Ile39) (Fig. 4B). In addition, the
side chains of AB-Tyr95 and AB-Gln32 stack and
contribute to the hydrophobic core. Side chains of
seven polar or charged residues positioned on helices
a2 and a5 are involved in hydrogen bond formation
over Interface-I (Table 2).
The crystal packing interactions revealed a second
putative dimer interface that we call Interface-II
(Fig. 4A, subunits colored in light and dark coral). This
interface is also formed by a four-helix arrangement
comprising symmetry-related a1 and a5 helices. How-
ever, at this interface, it is the N-terminal part of a5
that is involved. The polar sides of the two symmetry-
related N-terminal parts of helix a5 are facing each
other, and hydrogen bonds and two salt bridges are
formed across the dimer interface (Table 2). Hydropho-
bic contacts also exist between symmetry-related resi-
dues AB¢-Phe14, AB¢-Leu15 and AB¢-Leu18, where B’
is a crystallographic symmetry-related copy of subunit
B (symmetry transformation: )x, y +1⁄ 2, )z +1⁄ 2)
(Fig. 4C). In this dimer, the hydrophobic residues bur-
ied in the core of Interface-I are surface exposed.
pisa is an interactive tool that can be used for explo-

ration of protein interfaces [33]. pisa analysis of the
two interfaces identified in FocB crystals suggested
that both dimer alternatives are stable in solution. The
solvation energy effects, D
i
G, of Interface-I and II were
calculated to )16.1 kcalÆmol
)1
and )8.1 kcalÆmol
)1
,
respectively. Furthermore, DG
diss
, which indicates the
free energy of assembly dissociation, was calculated to
8.3 kcalÆmol
)1
for Interface-I, and 1.5 kcalÆmol
)1
for
Interface-II. Contact areas of Interface-I and -II were
estimated to 907 A
˚
2
and 851 A
˚
2
, respectively. Com-
bined, the output data from pisa suggest that dimer
Interface-I is significantly more stable than dimer

Interface-II.
FocB and PapB bind in the minor groove of
double-stranded DNA
In a previous study, Xia et al. [34] obtained evidence
that PapB binds in the minor groove of DNA. This
minor groove-binding property makes the protein
unusual in the perspective of transcriptional activa-
tors. To assess how the FocB protein interacts with
DNA, we performed a electrophoretic DNA gel
mobility shift test as a competition assay between
FocB and distamycin, a minor groove-binding drug
[35] and methyl green, a major groove-binding drug
[36]. That distamycin could bind to DNA under the
conditions used was evident from the mobility shift
observed when ‡ 4 lm of distamycin was added to the
DNA in the absence of protein (Fig . 5A, lane 4). At
lower concentrations, partial occupancy of distamycin
did not shift DNA (lanes 2 and 3). The DNA mobil-
ity shift caused by 125 nm of FocB (lane 5) was com-
pletely abolished when distamycin was present at
concentration of 4 lm or higher (lanes 9–10). Addition
of 1, 2, or 3 lm distamycin resulted in a gradually
reduced amount of FocB bound to the DNA (lanes
6–8). Combined, the results suggest that distamycin
competes with FocB for DNA-binding, and that the
FocB-binding site on the DNA was increasingly occu-
pied by distamycin at the tested concentrations. When
challenging the FocB–DNA complex formation with
methyl green at concentrations up to 100 lm,we
observed no apparent inhibition of the DNA-binding,

suggesting that FocB does not interact with the major
groove (Fig. 5B). When methyl green was added at
concentrations up to 100 lm in the absence of the
protein, we did not observe any shift in mobility of
the DNA but, at a very high concentration (1 mm),
the DNA did not enter the gel (data not shown). That
methyl green could bind to DNA under the conditions
used was evident from DNA-binding tests with CRP
(i.e. the cAMP receptor protein). CRP is a well char-
acterized, major groove-interacting, DNA-binding pro-
tein, used here as a control. The addition of 100 lm
methyl green abolished all of the CRP binding to
Table 2. Hydrogen bonds formed at the two plausible dimer inter-
faces of FocB.
Interface-I
Chain A Distance (A
˚
) Chain B
Glu 31 (Oe2) 2.5 Tyr 95 (Og)
Gln 32 (Ne2) 3.0 Tyr 96 (Og )
Gly 38 (O) 2.6 Arg 84 (Ng2)
Arg 84 (Ng2) 2.9 Gly 38 (O)
Asn 86 (Nd2) 2.6 Tyr 96 (Og)
Tyr 96 (Og) 3.0 Gln 32 (Ne 2)
Tyr 96 (Og) 2.6 Asn 86 (Nd2)
Interface-II
Chain A Distance (A
˚
) Chain B¢
Ser 76 (Oc) 3.2 Ser 76 (Oc)

Ser 76 (Oc) 3.3 Asn 72 (Nd2)
Asp 12 (Od1) 2.9 Arg 81 (Ne)
Asp 12 (Od2) 3.0 Arg 81 (Ng2)
Arg 81 (Ne) 2.8 Asp 12 (Od1)
Arg 81 (Ng2) 2.9 Asp 12 (Od2)
U. W. Hultdin et al. Structure of FocB
FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS 3373
DNA (data not shown). Taken together, our results
strongly suggest that FocB binds DNA by minor
groove interactions.
The FocB structure shows a fold similar to
DNA-binding HTH proteins
Structural similarity searches using the dali server
[37,38] identified a number of structures similar to
FocB. The top seven dali hits are presented in
Table 3. Generally, the protein structures identified by
dali comprise DNA-binding proteins with HTH
motifs and, for many of the hits, structures in complex
with DNA or RNA are available in the PDB. Struc-
tural comparisons of FocB with some of the top DALI
hits are shown in Fig.6.
The dali server rates structural similarity by the
z-score, where values above 2 are considered to be
significant hits. KorA, with a z-score above 7 and a
sequence identity of 18%, can tentatively be considered
relevant [38]. The secondary structure organization of
KorA with a recognition helix of a HTH motif flanked
by three short helices is also strikingly similar to the
FocB structure (Fig. 6A).
Furthermore, KorA is a homodimeric repressor pro-

tein with two HTH motifs that bind in the major
groove on opposite sides of the DNA [39]. With the
N-terminal end of its recognition helix, KorA recog-
nizes and binds a 12 bp symmetric operator. The side
chains of Gln37 in the first helix of the HTH motif,
and Arg48, Gln53 and Arg57 in its second helix, are
particularly important for specific interaction with the
DNA [39]. In FocB, these residues correspond to
Arg61, positioned on helix a4, and Asn72, Thr77 and
Arg81, positioned on a5. Interestingly, substitution of
Arg61 and Arg81 impair DNA-binding in PapB, indi-
cating that these residues are critical for DNA-binding
also in FocB [25].
The DNA-recognition domain of RNA polymerase
r
E
-factor, another of the FocB structurally similar
proteins, shows a DNA interaction similar to that of
KorA. The r
E
-factor is also a homodimer, with one
HTH motif per subunit, interacting exclusively with
the major grooves of two different DNA strands [40]
(Fig. 6B). Hepatocyte nuclear factor-1 (HNF-1b) also
shares structural similarity to FocB (Fig. 6C). Differ-
ent from KorA and the RNAP r
E
-factor, this protein
binds to the major groove using both helices of its
HTH motif [41]. Also, DNA bound to HNF-1b is ori-

ented  90° with respect to the position of the DNA
bound to KorA or RNAP r
E
-factor.
Among the top DALI-hits, we also found the struc-
ture of the G1 ⁄ S specific cyclin-D1 protein. In this
protein, the two helices that share structural similarity
to a HTH motif do not have DNA-binding function
A
B
Fig. 5. Gel mobility shift assay of the FocB protein binding to DNA
in the absence or presence of the DNA-binding compounds distamy-
cin (minor groove-binding) and methyl green (major groove-binding),
respectively. A 401 bp DNA fragment containing four repeats of the
9 bp long sequence constituting the primary FocB-binding sequence
[5] was used as target DNA. The shifted bands in the gel represent-
ing different DNA complexes with protein or the tested compounds
are indicated along the left side. (A) Effect of distamycin on FocB-
binding. (B) Lack of effect of methyl green on FocB-binding.
Table 3. DALI search results.
Protein Chain PDB code z-score rmsd Alignment length % sequence identity
KorA, transcriptional repressor protein B 2W7N 7.7 2.0 68 18
Hel308, helicase A 2P6U 5.8 2.6 73 15
G1 ⁄ S specific cyclin-D1 A 2W9Z 5.4 3.0 72 10
Hypothetical UPF0122 A 1XSV 5.1 2.8 64 6
RNA polymerase r
E
-factor D 2H27 4.9 2.6 60 12
HNF-1b B 2H8R 4.7 3.5 67 6
Signal recognition particle, M-domain A 1HQ1 4.7 2.6 60 5

Structure of FocB U. W. Hultdin et al.
3374 FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS
[42]. Other structures, such as domain 4 of the helicase
Hel308 (Fig. 6D) and the RNA-binding M-domain of
Ffh (Fig. 6E), gave examples of nucleic acid interac-
tions different from classical HTH–major groove inter-
actions. Domain 4 of Hel308 consists of a seven-helix
bundle and together with other domains it forms a
ring around the 3¢ tail of the unwound DNA oligonu-
cleotide [43]. In this interaction, the central helix of
domain 4 (corresponding to a5 in FocB) provides a
ratchet for directional transport of the product DNA
tail across the protein. Arg592 and Trp599 in the
ratchet helix, corresponding to Thr77 and Arg84 in
FocB, stack on base moieties of the single-stranded
DNA. Amino acids that are positioned in the helix
corresponding to helix a4 in FocB are not involved in
DNA interaction. Ffh is a protein constituent of the
signal recognition particle [44] and its M-domain con-
tains a HTH motif that binds to the minor groove of
signal recognition particle RNA with the small first
helix of the motif (analogous to a4 in the FocB struc-
ture) (Fig. 6E).
Residues important for oligomerization and
DNA-binding
Alanine substitutions in PapB, made at positions con-
served throughout the PapB family, have revealed a
number of specific amino acids that appear to be par-
ticularly important with respect to the ability of pro-
teins to bind to their target DNA, and for their ability

to form oligomeric complexes [25,34]. Two residues
found to be important for DNA-binding in PapB (i.e.
Arg61 and Cys65) are conserved and located on helix
a4 in FocB (i.e. the helix preceding the presumed rec-
ognition helix, a5) (Fig. 3). PapB binds DNA in an
oligomeric fashion, probably in the form of dimers or
tetramers [34]. Conserved residues shown to be impor-
tant for oligomerization in PapB are spread out over
the FocB structure. Some of these residues (i.e. Asp53,
A
B
C
D
E
Fig. 6. Protein–DNA and –RNA complex structures of the top five
structural homologs of FocB identified by
DALI [38]. Ribbon repre-
sentations are shown to the left and overlays of Ca-traces of the
structurally similar proteins (light blue) and FocB (dark red) are
shown to the right. DNA strands are shown in yellow and orange,
and the RNA strand is shown in coral. Parts of the structures
superimposing on the HTH motif of FocB are highlighted in blue in
the ribbon representation. To visualize the variety of DNA ⁄ RNA-
binding sites, all proteins are shown with the same orientation of
their HTH motif. (A) KorA (PDB code 2W7N), chain B, residues 3–
97 (38–67 in blue). (B) RNA polymerase r
E
-factor (PDB code 2H27),
chain D, residues 122–190 (185–196 in blue). (C) HNF-1b (PDB
code 2H8R), chain B, residues 90–185 (157–184 in blue). (D)

Hel308 (PDB code 2P6R), chain A, residues 508–612 (576–612 in
blue). (E) Ffh M-domain (PDB code 1HQ1), chain A, residues 13–
83, (48–82 in blue).
U. W. Hultdin et al. Structure of FocB
FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS 3375
Tyr54, Leu55 and Val56) are located in helix a3.
Because this helix appears necessary for stabilizing the
HTH motif, which is in itself apparently insufficient
for independent folding, these mutations most likely
affect the conformation of the whole protein structure.
Other residues important for oligomerization include
residues Leu35 and Leu36, which are buried in the
dimer Interface-I. In this dimer constellation, the side
chains of residues Tyr74, Phe75 and Ser76, also previ-
ously shown to be important for oligomerization, are
exposed on the surface of the protein. These residues,
however, are buried at Interface-II.
Discussion
The FocB protein is a transcription factor involved in
the regulation of genes for production of F1C fimbriae
that are commonly expressed by UPEC. FocB belongs
to the PapB family of adhesin regulators. Within this
family, PapB is currently the most thoroughly charac-
terized member and shares 81% sequence identity with
FocB. The most significant difference in their
sequences is located at the N-terminal ends of the pro-
teins, a region that is predicted to form a b-strand in
the PapB protein (Fig. 2). In the present study, we
have structurally characterized residues 10–99 of the
109 amino acid residue protein FocB at 1.4 A

˚
resolu-
tion. The structure comprises five a-helices, including a
HTH motif that is quite common for many DNA-
binding proteins. From the amino acid sequence alone,
no obvious recognition motifs similar to other DNA-
binding proteins could be identified in FocB. Compar-
ing the PapB sequence with the FocB structure shows
that differences in their sequences are predominantly
located in the a1-loop-a2 region of the N-terminus as
well as in the C-terminal part of the two proteins.
Thus, the core structure of PapB is very likely identical
to that of FocB.
Results from DNA-shift assays suggest that FocB,
similar to PapB, is a minor groove-binding protein. In
general, minor groove-binding proteins show various
degrees of sequence specificity when binding to DNA.
The lack of unique chemical features present within
the minor groove requires various strategies for recog-
nition. For example, the TATA box-binding protein
finds and interacts with the minor groove of the TATA
element, onto which it binds analgous to a saddle on a
horse [45,46]. The integration host factor, on the other
hand, uses a so-called winged helix motif to bind to
regions of unusually narrow minor grooves [47]. A
common feature of minor groove-binding proteins is
their ability to bend DNA. For TATA box-binding
protein and integration host factor, intercalation of
hydrophobic residues between base pair steps in the
DNA is the main cause of the extreme DNA-bending

ability of these proteins [48]. Also, LacI [49,50] and
PurR [51], members of the LacI family, bend their
operator DNA. Both LacI and PurR form dimers
attached tail-to-tail, where each dimer consists of an
N-terminal DNA-binding domain and a C-terminal
oligomerization domain. The DNA-binding headpieces
of the N-terminal domains contain conventional major
groove-binding HTH motifs, but symmetric hinge
a-helices immediately adjacent to the headpieces bind
deep in the minor groove, and intercalate leucine resi-
dues into the central base pair step, which cause the
DNA to bend.
The structure of the FocB subunit contains one
HTH motif. Generally, this motif is in its smallest
functional form part of a core of three helices that
form a right-handed helical bundle with a partly open
configuration. The HTH motif, as it is known from
transcription factors in both prokaryotes and eukary-
otes, recognizes a specific DNA-sequence and is mostly
associated with major groove-binding. Through inter-
action between the second helix of the HTH motif (i.e.
the recognition helix) and the DNA major groove, the
sequence information is accessible for the protein
[32,52,53]. Recently, proteins with minor groove-bind-
ing HTH motifs have been identified. One example is
the human DNA repair protein O6-alkylguanine-
DNA-alkyltransferase [54,55]. Because DNA damage
is not sequence dependent, it is favorable for DNA
repair, as well as other types of nucleotide-flipping
proteins, not to bind specifically to DNA. In O6-alkyl-

guanine-DNA-alkyltransferase, one arginine residue,
which is centrally positioned on the recognition helix,
stacks between DNA bases in the minor groove, caus-
ing the DNA to bend. In addition, small hydrophobic
residues of the recognition helix are presented to the
minor groove of the DNA, providing a nonsequence-
specific interaction [54].
FocB and PapB bind to the minor groove of their
target DNA. This is in agreement with their ability to
bind A ⁄ T-triplets occurring at 9 bp intervals [34],
where a classical, direct major groove interaction is less
expected. We found that the conserved residues Arg61
and Cys65, known to be important for DNA-binding,
are surface exposed and located in the helix preceding
the putative recognition helix (Fig. 3). Also in the
major groove-binding protein KorB, side chains that
are not located in the recognition helix have been
shown to be essential for the binding specificity [56]. In
that case, the specific Thr211 and Arg240 are situated
outside of the HTH motif, whereas the HTH itself is
typically placed with the recognition helix running
Structure of FocB U. W. Hultdin et al.
3376 FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS
along the major groove. Taken together, our findings
suggest that the HTH motif in FocB is involved in
DNA interactions that very likely are different from
the classical HTH–major groove contacts.
From dali searches, several proteins with similar
structures to FocB were identified. We tried to identify
the DNA-binding site in FocB based on the proteins

identified by dali; however, the latter proteins dis-
played very different types of DNA or RNA interac-
tions (Fig. 6). Therefore, at this point, we refrain from
speculating on where the exact DNA-binding site of
FocB.
FocB and PapB bind to DNA in an oligomeric fash-
ion [34]. From the crystal structure of FocB, two pos-
sible dimeric arrangements were identified. Of these,
Interface-I is significantly more hydrophobic. Further-
more, one of the dali hits, the hypothetical
DNA-binding UPF0122 protein SAV1236 from
Staphylococcus aureus (PDB code 1XSV; unpublished)
(Table 3), is a dimer with a packing interaction
resembling that of Interface-I in FocB (Fig. 7).
Mutants that impair PapB oligomerization [25] are
localized both at Interface-I and -II. We therefore con-
sider that Interface-I represents the most stable dimeric
form of FocB, but that Interface-II might play a role
in the formation of larger oligomeric structures neces-
sary for DNA-binding [34]. We hypothesize that
several FocB dimers can bind to DNA side by side.
The crystal structure of the FocB transcription
factor provides an important starting point for further
analyses aiming to understand the mechanisms of
fimbrial gene regulation at the molecular level not
just for FocB, but also for the entire family of related
proteins.
Experimental procedures
Protein expression and purification of native and
Se-Met FocB

The overexpression and purification of native FocB (109
amino acid residues) has been described previously [27].
The full-length FocB protein was cloned into pETM11 and
overexpressed in E. coli strain BL21 (DE3). The protein
was purified on a Ni-NTA (Qiagen, Valencia, CA, USA)
column followed by a Superdex 75 column (GE Healthcare,
Milwaukee, WI, USA). During purification, the 6-His tag
was removed with tobacco etch virus protease, leaving two
extra residues, Gly and Ala, followed by Met1 correspond-
ing to the native N-terminus. Pure fractions of the protein
in 10 mm Hepes (pH 7.9), 500 mm NaCl, 5 mm EDTA and
0.1% b-mercaptoethanol were pooled and concentrated to
16 mgÆmL
)1
, filtered through a 0.2 lm filter and stored at
4 °C.
Selenomethionyl labeling of FocB (Se-Met FocB) was
performed using the protocol as described previously [57].
FocB Se-Met was overexpressed in E. coli BL21 (DE3) cells
grown in minimal medium (48 mm Na
2
HPO
4
,22mm
KH
2
PO
4
,9mm NaCl, 19 mm NH
4

Cl, 2 mm MgSO
4
, 0.1 mm
CaCl
2
,4gÆL
)1
glucose) with the addition of kanamycin
(0.1 mgÆmL
)1
)at37°C until D
600
= 0.6. The temperature
AB
Fig. 7. Structural similarity between (A) the
dimer Interface-I of FocB and (B) the dimer
interface of protein UPF0122 (PDB code
1XSV, unpublished). Residues Leu13-Leu76
of the protein matched residues Ser27-
Ala93 of FocB with a z-score of 5.1
(Table 2). The structures are shown in two
orientations. The visual alignment is based
on the position of the HTH motif.
U. W. Hultdin et al. Structure of FocB
FEBS Journal 277 (2010) 3368–3381 ª 2010 The Authors Journal compilation ª 2010 FEBS 3377
was lowered to 25 °C and amino acids (lysine 100 mgÆL
)1
,
threonine 100 mgÆL
)1

, phenylalanine 100 mgÆL
)1
, leucine
50 mgÆL
)1
, isoleucine 50 mgÆL
)1
, valine 50 mgÆL
)1
, proline
50 mgÆL
)1
) and seleno-l-methionine 50 mgÆL
)1
(Sigma,
St Louis, MO, USA) were added. After 15 min, the culture
was induced overnight with isopropyl thio-b-d-galactoside,
100 mgÆL
)1
culture. The culture was harvested by centrifu-
gation at 53 000 g for 30 min. Purification of Se-Met-FocB
was performed in the same way as described for the native
protein [27]. Pure fractions of the protein in 10 mm Hepes
(pH 7.9), 500 mm NaCl, 5 mm EDTA and 0.1% b-mercap-
toethanol were pooled and concentrated to 13.5 mgÆmL
)1
and stored at 4 °C.
Crystallization
Both native [27] and Se-Met FocB were crystallized by
hanging-drop vapor diffusion of a mixture of equal vol-

umes of protein solution and well solution. The well solu-
tion contained typically 0.1 m Mes (pH 6.5) and 1.5–1.75 m
MgSO
4
. Crystals grew at 22 °C to dimensions of 0.1 · 0.1
· 0.05 mm within 2 weeks.
X-ray data collection and processing
Diffraction data from one Se-Met FocB crystal were
recorded at 100 K at three wavelengths corresponding to
the peak (k = 0.97920 A
˚
), the inflection point (k =
0.97945 A
˚
) and a high-energy remote (k = 0.97535 A
˚
)
wavelength of a selenium K-edge absorption profile on
beamline ID23-1 at ESRF (Grenoble, France). A total of
180 frames of data with an oscillation angle of 1° were col-
lected at each wavelength. The exposure time was 0.3 s per
frame.
Native data were collected to a resolution of 1.4 A
˚
.
A total of 360 frames of data with an oscillation angle of
0.45° were collected. The exposure time was 0.5 s per
frame. All datasets were processed with xds [58]. The native
data set was scaled with scala from the ccp4 software suite
[59].

Structure determination and refinement
The Se-Met FocB structure was solved using the three-
wavelength multiwavelength anomalous diffraction protocol
of auto-rickshaw: the EMBL-Hamburg automated crystal
structure determination platform [60]. The input diffraction
data were converted for use in auto-rickshaw using soft-
ware from ccp4 [59]. The ARP ⁄ wARP module for tracing
helices and strands in auto-rickshaw correctly built 84 of
the protein’s 109 amino acid residues. Manual map inspec-
tion and model building were performed with coot [61,62]
and positional refinement was performed with refmac5
[59], using the maximum likelihood residual, anisotropic
scaling, bulk-solvent correction and atomic displacement
parameter refinement [63], as well as the translation, libra-
tion, screw-rotation method [64]. Throughout the refine-
ment, the protein subunits were treated independently and
all modeled residues were refined with single conformations
and occupancies set to 1.
Structure validation was performed with coot [61,62]
and beaverage [59]. The calculation of contact surface
areas between molecules was performed with pisa [33].
Images were prepared with ccp4mg [59]. Homology
searches were performed with dalilite, version 3 [37,38].
Analysis of protein–DNA-binding properties
Gel mobility shift assays were performed to determine the
DNA-binding properties. DNA fragments containing the
FocB-binding site were obtained by PCR using oligonucleo-
tides yielding the 401 bp foc I DNA fragment [5]. Plasmid
pBSN50 was used as a template for foc I DNA [23]. FocB
protein (125 nm) was mixed with the foc I DNA fragments

in the presence of 50 mm KCl in buffer B [25 mm Hepes
(pH 7.5), 0.1 mm EDTA, 5 mm dithiothreitol, 10% glyc-
erol] in a final volume of 10 lL), together with the indi-
cated amounts of distamycin or methyl green as described
previously [34]. The amount of DNA was 125 ng in the
10 lL assay mixture. The reaction mixtures were incubated
at 37 °C for 15 min and then immediately loaded onto a
Novex 6% DNA Retardation gel (Invitrogen, Carlsbad,
CA, USA) for electrophoresis. The gel was subsequently
stained with GelRed Nucleic Acid Gel Stain (Biotium, Inc.,
Hayward, CA, USA) for 30 min then visualized using the
Quantity One ChemiDoc system (Bio-Rad, Hercules, CA,
USA).
Acknowledgements
We thank the European Synchrotron Radiation Facil-
ity (Grenoble, France) for provision of the synchrotron
radiation facilities, as well as Drs Uwe H. Sauer, Gun-
ter Stier, Anders O
¨
hman and Michael Murphy for
their valuable discussions and critical reading of the
manuscript. The work was performed within the Umea
˚
Centre for Microbial Research (UCMR) and was sup-
ported by the Swedish Research Council, project
K2010-68X-13001-12-3 and project 621-2005-3151.
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