Tài liệu Báo cáo khoa học: Structural and biochemical characterization of a human adenovirus 2/12 penton base chimera pptx

Structural and biochemical characterization of a human
adenovirus 2/12 penton base chimera
Chloe Zubieta
1,
*, Laurent Blanchoin
2
and Stephen Cusack
1
1 European Molecular Biology Laboratory, Grenoble Outstation, France
2 Laboratoire de Physiologie Cellulaire Vegetale, Commissariat a l’Energie Atomique, Centre National de la Recherche Scientifique,
Institut National de la Recherche Agronomique, Universite Joseph Fourier, Unite Mixte de Recherche 5168, Grenoble, France
Adenoviruses are nonenveloped double-stranded DNA
viruses found in mammalian and non-mammalian
vertebrates. Human adenoviruses are divided into six
subgroups (A–F) based on genetic organization, hema-
gluttination patterns, immuno-crossreactivity, and nuc-
leotide content. Over 50 serotypes have been identified
in humans; these cause generally mild respiratory,
enteric and ocular disease. However, in immunocom-
promised, very young, or elderly individuals, adenoviral
infections can lead to serious illness or death [1,2].
Apart from their role as a common human pathogen,
adenoviruses are one of the most studied vectors for
gene delivery due to extensive knowledge of their
biology and the ability to manipulate the adenoviral
genome [3–5].
The adenoviral T ¼ 25 icosahedral capsid consists
of three major polypeptides: the trimeric hexon, which
forms the facets of the particle, the pentameric penton
base (pb), which forms the vertices, and the trimeric
fiber protein, which extends from the penton base at

the vertex positions. Additionally, cementing proteins
such as IIIa, VI, VIII and IX help stabilize the capsid,
reinforcing the penton–hexon and hexon–hexon inter-
actions. The capsid contains 720 copies of the hexon
Keywords
adenovirus; crystal structure; fiber;
fluorescence anisotropy; penton
Correspondence
C. Zubieta, SLAC ⁄ SSRL, 2575 Sandhill
Road, Menlo Park, CA 94025, USA
Fax: +1 650 926 3292
Tel: +1 650 926 2992
E-mail:
*Present address
Stanford Synchrotron Radiation Facility,
Menlo Park, CA, USA
(Received 6 June 2006, revised 11 July
2006, accepted 27 July 2006)
doi:10.1111/j.1742-4658.2006.05430.x
The vertex of the adenoviral capsid is formed by the penton, a complex of
two proteins, the pentameric penton base and the trimeric fiber protein.
The penton contains all necessary components for viral attachment and
entry into the host cell. After initial attachment via the head domain of the
fiber protein, the penton base interacts with cellular integrins through an
Arg-Gly-Asp (RGD) motif located in a hypervariable surface loop, trigger-
ing virus internalization. In order to investigate the structural and func-
tional role of this region, we replaced the hypervariable loop of serotype 2
with the corresponding, but much shorter, loop of serotype 12 and com-
pared it to the wild type. Here, we report the 3.6 A
˚

crystal structure of a
human adenovirus 2 ⁄ 12 penton base chimera crystallized as a dodecamer.
The structure is generally similar to human adenovirus 2 penton base, with
the main differences localized to the fiber protein-binding site. Fluorescence
anisotropy assays using a trimeric fiber protein mimetic called the minifiber
and wild-type human adenovirus 2 and chimeric penton base demonstrate
that fiber protein binding is independent of the hypervariable loop, with a
K
d
for fiber binding estimated in the 1–2 lm range. Interestingly, competi-
tion assays using labeled and unlabeled minifiber demonstrated virtually
irreversible binding to the penton base, which we ascribe to a conforma-
tional change, on the basis of comparisons of all available penton base
structures.
Abbreviations
hAd, human adenovirus; MPD, 2-methyl-2,4-pentane diol; NCS, noncrystallographic symmetry averaging; pb, penton base; TMR,
tetramethylrhodamine.
4336 FEBS Journal 273 (2006) 4336–4345 ª 2006 The Authors Journal compilation ª 2006 FEBS
and 60 copies of the penton base monomers. In addi-
tion to its role as a critical component of the viral cap-
sid, the penton base also contains an Arg-Gly-Asp
(RGD) motif that acts as a trigger for endocytosis of
the adenovirus into the host cell. Furthermore, the
architecture of the entire virus, including the positions
of the cementing proteins, has been revealed in detail
by recent high-resolution cryoelectron microscopy
reconstructions [6–11].
Initial cell attachment occurs via interactions of the
fiber protein C-terminal head domain. For the major-
ity of serotypes, including adenoviruses 2 and 12, the

Coxsackie- and adenovirus receptor (CAR), an integral
membrane protein, shown to be a component of the
tight junction in epithelial cells [12], is the primary cel-
lular receptor. Upon virus attachment, the RGD motif
of the penton base binds to a
V
b
3
, a
V
b
5
,ora
V
b
1
inte-
grin, which acts as a secondary receptor [13–15]. This
interaction of the RGD motif with the host cell causes
clustering of integrins and activates a signaling cas-
cade, allowing viral entry into clathrin-coated pits and
endosomes [14,16]. It is thought that upon integrin
binding and before endocytosis, the fiber protein is
shed from the virus [13,17–19]. One hypothesis for the
mechanism of fiber removal from the virus involves a
conformational change triggered by the penton base
binding to integrin. The process by which integrin
binding may be coupled to structural changes in the
penton base, allowing fiber release, is not clear, but
could involve changes in the hypervariable loop upon

integrin binding being communicated to the fiber-bind-
ing region of the penton base.
Although the adenovirus penton base exhibits a high
degree of sequence homology (typically over 70%
homology between different subgroups of human
adenoviruses), the RGD motif is located in a hyper-
variable loop region of the penton base protein. This
region can vary in length from approximately eight
residues in human adenovirus serotype 12 (hAd12) to
over 70 residues in human adenovirus 2 (hAd2). Previ-
ous crystallographic studies of the hAd2 penton base
revealed this region to be very flexible, with the hyper-
variable loop almost completely disordered. At a
unique crystal contact, a helix turn from the hypervari-
able loop was identified; however, the amino acid
sequence could not be definitively assigned [20]. Cryo-
electron microscopy reconstructions of adenovirus 2
[21] with an RGD-recognizing antibody and with a
soluble a
V
b
5
integrin [16] show only diffuse density for
the hypervariable loop region; however, the resolution
was limited to  21 A
˚
. In contrast, cryoelectron micro-
scopy reconstructions of complexes with soluble inte-
grin and hAd12 gave more defined density in the
region of the RGD motif due to the better order of

the shorter hypervariable loop in this serotype [16].
Higher-resolution cryoelectron microscopy reconstruc-
tions (12 A
˚
) of dodecahedral penton and penton base
particles formed by the hAd3 serotype revealed addi-
tional density in the region of the hypervariable loop,
implying some favored conformations of the region
[22]. The electron microscopy data for serotypes hAd3
and hAd12 and the crystallographic data for hAd2
raise the possibility that the hypervariable loop could
possess secondary structural elements potentially
important for integrin recognition or binding.
We constructed an hAd2pb ⁄ 12pb chimera by repla-
cing the long hypervariable loop ( 73 residues) of
hAd2pb with the short hypervariable loop ( 8 resi-
dues) of hAd12pb. Note that the sequence identity
between hAd2 and hAd12 penton bases outside the
hypervariable region is approximately 78%. We hypo-
thesized that the shorter hypervariable loop of hAd12
would be more structured relative to the longer hAd2
hypervariable loop and allow for crystallographic
characterization of this region of the penton base.
Additionally, the chimeric construct would enable us
to assess its possible role in the binding of the fiber
protein in serotypes such as hAd2, which contain a
long hypervariable region. The long hypervariable
loop could directly interact with the fiber protein, but
the shorter hAd12 hypervariable loop would be unable
to reach the fiber protein-binding site. Thus, by dra-

matically shortening the hypervariable loop to the
hAd12 sequence, we eliminated any possible direct
interactions between the hypervariable loop of the
penton base and the fiber protein. We present here the
3.6 A
˚
crystal structure of an hAd2pb ⁄ 12pb chimera
and fluorescence anisotropy binding data of the wild
type and the chimeric construct with a fiber protein
mimetic.
Results and Discussion
The 73-residue hypervariable loop of hAd2pb was
replaced with the eight-residue hypervariable loop
from hAd12 (Fig. 1). Owing to the proteolytic sensi-
tivity of the construct, a more stable N-terminally
truncated (49-TGGR ) version was designed. All
structural studies presented here used the ) 49 N-ter-
minally truncated hAd2pb ⁄ 12pb chimera.
Analogous to the hAd2 penton base, the hAd2 ⁄ 12
chimeric construct formed well-behaved pentamers in
solution, as confirmed by gel filtration and negative-
stain electron microscopy (data not shown). As with
hAd2pb, the solvent environment has marked effects
on the equilibrium between pentamer and dodecamer
C. Zubieta et al. Human adenovirus 2 ⁄ 12 penton base chimera
FEBS Journal 273 (2006) 4336–4345 ª 2006 The Authors Journal compilation ª 2006 FEBS 4337
(regular arrangements of 12 pentamers), and the chi-
mera formed dodecahedral particles under crystalliza-
tion conditions. These crystallization conditions were,
however, different from those for hAd2. For hAd2pb,

high concentrations of ammonium sulfate (1.6 m) and
dioxane (10%) were necessary for dodecahedra forma-
tion [20], whereas hAd2 ⁄ 12pb crystallized in the
dodecahedral form in high (50% v ⁄ v) 2-methyl-2,4-
pentane diol (MPD) solvent conditions. The ionic
strength of the solvent environment and ⁄ or presence
of small organic compounds (dioxane and MPD)
seems to favor formation of dodecahedra over isolated
pentamers. Previous structural analysis of the residues
at the penton–penton interface of the hAd2pb dodeca-
hedron showed that there is no obvious pattern char-
acterizing the penton bases of those serotypes which
do or do not form dodecahedra during adenoviral
infection [20]. Indeed, we suspect that all penton bases
are capable of forming dodecahedra, depending on
two factors: first, the solvent conditions affecting the
dodecamer–pentamer equilibrium, and second, proteo-
lysis or truncation of the penton base N-terminus. It
has recently been shown that formation of hAd3
dodecahedra is favored by truncation of the N-ter-
minal region [16], and crystals of the hAd2 and
hAd2 ⁄ 12 chimera dodecahedra were obtained with
protein N-terminally truncated by 49 residues [19].
The structure shows that the truncated N-terminus of
the protein faces the interior of the particle, and small
solvent channels between pentamers are not extensive
enough to provide egress for the N-termini from the
particle while retaining the dodecahedral structure.
Calculations of the interior volume of the dodeca-
hedral particle based on a cavity radius of 40 A

˚
in hAd2 ⁄ 12pb and hAd2pb give a volume of
 300 000 A
˚
3
, too small to accommodate an extra
2940 residues corresponding to the N-terminal exten-
sion from the 60 copies of the full-length protein. The
formation of small subviral particles by truncated viral
capsid proteins is not limited to adenovirus. For exam-
ple, this phenomenon of spontaneous assembly into
regular particles has been noted for recombinant
human papilloma virus L1 protein, a pentameric cap-
sid protein. Only upon a 10-residue N-terminal trunca-
tion does the L1 protein form small virus-like particles
of icosahedral symmetry [23].
The physiologic role, if any, of penton base dode-
camer formation in infected cells has not been defined
for adenovirus. However, the N-terminal extremity of
the penton base contains two PPxY motifs, shown to
interact with the WW domains of cellular ubiquitin
ligases [24,25]. As these motifs are presumably import-
ant for adenovirus infection, it would be undesirable
to incorporate truncated penton bases into virions,
and dodecahedron formation could be a mechanism to
avoid this.
Fig. 1. Sequence alignment of adenovirus penton base. hAd2 (accession number P03276), hAd12 (accession number P36716) and the
hAd2 ⁄ 12 chimera are aligned with the consensus secondary structure above the sequence. b-Strands are in orange and helices are in green.
The hypervariable loop is highlighted in yellow, with the RGD motif in red.
Human adenovirus 2 ⁄ 12 penton base chimera C. Zubieta et al.

4338 FEBS Journal 273 (2006) 4336–4345 ª 2006 The Authors Journal compilation ª 2006 FEBS
Overall structure
The hAd2⁄ 12pb construct crystallized in space group
I222 with unit cell dimensions a ¼ 266.50, b ¼ 292.92,
and c ¼ 307.30, and three pentamers (1 ⁄ 4 dodecahed-
ron) per asymmetric unit (Table 1). The complete do-
decahedral particle of 12 pentamers was formed by the
space group symmetry operators (Fig. 2). The original
hAd2pb, with a full-length hypervariable loop, crystal-
lized in a C2 cell with one dodecahedron per asymmet-
ric unit, but exhibited a similar packing arrangement
to the chimeric protein [20]. The structure was phased
by molecular replacement using the hAd2pb pentamer
as a model and searching for three copies in the asym-
metric unit. Initial maps were improved by rigid body
refinement followed by 15-fold noncrystallographic
symmetry averaging (NCS) of the electron density.
Owing to the high degree of NCS, the electron density
maps were of high quality and readily interpretable.
The hAd2 ⁄ 12pb forms a stable pentamer under
physiologic conditions. Pentamer formation involves
contacts throughout the polypeptide chain and hides a
large amount of the hydrophobic surface area. Surface
area calculations for the pentamer give a total surface
area of  81 000 A
˚
2
with 30% ( 24 000 A
˚
2

) as con-
tact area. Thus, a large amount of the available surface
area of the molecule is buried upon pentamerization,
increasing the stability of the protein.
The hAd2 ⁄ 12 penton base monomer can be divided
into a basal jellyroll domain formed by two b-sheets
and a distal domain formed by insertions between the
strands of the jellyroll domain (Fig. 3). Antiparallel
b-sheets made up of strands CHEF and BIDG pack
against each other, forming the jellyroll topology,
a typical viral capsid protein fold [26]. The distal
domain is formed by an insertion of  230 residues
(residues 133–367) between strands D and E and a
smaller insertion of  50 residues (residues 404–458)
between strands F and G. The hypervariable loop,
Table 1. Data collection and refinement statistics for hAd2/12 pen-
ton base.
Ad2 ⁄ 12 penton base
chimera
Data collection statistics
Space group I222
Cell dimensions (A
˚
) a ¼ 266.50
b ¼ 292.92
c ¼ 307.30
Oscillation range 0.5°
Contents of asymmetric unit 3 pentamers
Observations
Total measured reflections 363 715

Unique reflections 124 622
Resolution (A
˚
) 19.8–3.6
Completeness
a
(3.69–3.6) 0.90 (0.92)
R
factor
a,b
0.13 (0.64)
I ⁄ r 4.12 (1.33)
Average measurement redundancy 2.9
NCS redundancy 15-fold
Refinement statistics
Resolution (A
˚
) 15.0–3.6
Working reflections 115 706
Test reflections 3041
Number of nonhydrogen atoms 53 065
NCS averaging statistics
c
Correlation ⁄ R
fac
d
0.90 ⁄ 0.32
Model refinement R
cryst
⁄ R

free
(%)
e
27.5 ⁄ 32.8
Model geometry
RMSD bond lengths (A
˚
) 0.015
RMSD bond angles (°) 1.68
Ramachandran plot
Favored + additional regions 96.7%
Disallowed (number) 3.3% (3)
a
Values in parentheses refer to the highest resolution shell.
b
R
factor
¼ S|I ) <I> | ⁄SI,where<I> is the average value of a reflection,
I.
c
Averaging with RAVE to high resolution limit with 15-fold sym-
metry.
d
Correlation between the densities of all noncrystallographic
symmetry averaging (NCS)-related points. R
fac
¼ S|F
obs
) F
map

| ⁄
SF
obs
, where F
map
is the Fourier coefficient of the back-transformed
averaged map.
e
Tight NCS restraints were used for all backbone
atoms and relaxed NCS restraints were used for side chains during
final refinements. R
cryst
¼ S|F
obs
) F
calc
| ⁄SF
obs
, where summation
is over data used in the refinement. R
free
was calculated using
2.5% of the observed reflections excluded from refinement. Exclu-
ded data were randomly selected.
Fig. 2. Oligomerization of hAd2 ⁄ 12 penton base. (A) Monomer,
pentamer, and dodecamer. The orientation of the monomer in red
is kept throughout. (B) Asymmetric unit consisting of three pentam-
ers (left) and the full dodecamer formed by the space group sym-
metry (right). The pentamers in the asymmetric unit are in red.
C. Zubieta et al. Human adenovirus 2 ⁄ 12 penton base chimera

FEBS Journal 273 (2006) 4336–4345 ª 2006 The Authors Journal compilation ª 2006 FEBS 4339
containing the RGD motif, is located at the top of
the distal domain within the first insertion, and faces
the solvent-exposed exterior of the particle. As in the
hAd2pb structures, even the shorter Ad12 hypervaria-
ble loop is highly flexible, as demonstrated by relat-
ively poor electron density and high temperature
factors. In all monomers, residues 297–317 were disor-
dered and not modeled.
The second insertion into the jellyroll domain con-
tains part of the putative fiber protein-binding site.
This portion of the protein undergoes a conformation-
al change upon fiber protein binding, with helix 7
kinking almost 45° to form a binding cleft for the
N-terminus of the fiber protein [20]. Structural align-
ments of the hAd2 ⁄ 12pb chimera with the hAd2pb
fiber peptide bound and unbound structures reveal a
high degree of overall conservation (Figs 3B and 4).
Surprisingly, the hAd2 ⁄ 12pb structure most closely
resembles the fiber peptide-bound form of hAd2pb;
however, no fiber peptide was present during crystal-
lization, and the fiber-binding site is empty.
A possible explanation for the observed conforma-
tion of helix 7 is the effect of the solvent environment,
specifically high concentrations (50%) of MPD, favor-
ing a conformation mimicking the fiber protein-bound
state. HAd2pb and the hAd2pb ⁄ fiber peptide complex
were both crystallized from ammonium sulfate ⁄ diox-
ane solutions, not MPD solutions [20]. The use of high
concentrations of a small organic alcohol will affect

the solvent structure of the protein. Studies quantifying
the effects of MPD on protein structure have demon-
strated that the alcohol tends to bind in hydrophobic
sites, particularly at leucine side chains [27]. The fiber
Fig. 3. The hAd2 ⁄ 12 and wild-type hAd2 penton base monomers.
(A) Ribbon diagram of the hAd2 ⁄ 12 penton base monomer is in
blue (left) and rainbow (right). The N-terminus and C-terminus are
labeled in both monomers. On the left, the b-strands of the jellyroll
domain are labeled, with the BIDG sheets in yellow and the CHEF
sheet in cyan. (B) Left, ribbon diagram of the hAd2 penton base
(PDB code 1X9P) (green) with a portion of the hypervariable loop
(red) and helix 7 (blue). Middle, ribbon diagram of the hAd2 ⁄ 12 chi-
mera (green) and helix 7 (blue). Right, hAd2 fiber peptide complex
(PDB code 1X9T) with helix 7 in blue and the fiber peptide in
magenta.
Fig. 4. Comparison of the hAd2, hAd2 ⁄ 12 and hAd2–fiber peptide
complex structures. (A) Stereo overlay of hAd2 penton base (yel-
low), hAd2 ⁄ 12 (blue) and hAd2 fiber peptide complex (pink). The
hAd2 monomers are partially transparent, for clarity. The gray box
outlines helix 7. (B) Stereo overlay of helix 7 with hAd2 penton
base (yellow), hAd2 ⁄ 12 (blue), and hAd2 fiber peptide complex
(pink). The fiber peptide is drawn as ball-and-stick and colored by
atom.
Human adenovirus 2 ⁄ 12 penton base chimera C. Zubieta et al.
4340 FEBS Journal 273 (2006) 4336–4345 ª 2006 The Authors Journal compilation ª 2006 FEBS
protein-binding site consists primarily of hydrophobic
residues, including Leu193. It is possible that these
hydrophobic interactions lead to a conformational
change of helix 7. Although the modest 3.6 A
˚

resolu-
tion of the hAd2 ⁄ 12pb crystal structure precludes the
location of any ordered MPD molecules within the
fiber protein-binding site, this is a likely possibility.
A second hypothesis for the observed conformation
of helix 7 entails conformational coupling of the hyper-
variable loop to the fiber protein-binding region. It has
been shown that the fiber protein is shed from the virus
upon integrin binding by the penton base hypervariable
loop and most likely before endocytosis [13,17–19].
Upon addition of soluble RGD peptides that interfere
with penton base binding to integrin, the virion exhib-
ited impaired endocytosis. Interestingly, in this experi-
ment, the fibers were not shed from the virus [19].
These data raise the possibility of coupling of fiber
release to binding of the RGD motif of the hypervaria-
ble loop to cellular integrins. Alterations in hypervaria-
ble loop topology, in this case engendered by
shortening the loop to the Ad12pb sequence, could
have allosteric effects on the fiber-binding site. In order
to investigate this possibility, we performed fluores-
cence-based binding assays of the wild-type hAd2pb
and the hAd2 ⁄ 12 chimera with a fiber protein mimetic
to determine whether fiber binding or fiber release was
coupled to changes in the hypervariable loop.
Binding assays
Owing to difficulties in expressing and purifying the
full-length fiber protein, a 75 amino acid ‘minifiber’
construct was used as a fiber protein mimetic. Previous
structural studies of hAd2pb with a 22 amino acid

peptide from the N-terminus of the fiber protein have
mapped the fiber protein interaction region to a hydro-
phobic stretch of amino acids at the N-terminus of the
fiber protein [20]. In order to emulate the oligomeriza-
tion state of the fiber, a trimerization domain was
fused to an N-terminal fiber protein construct (gift of
G Nemerow). This construct consists of the first 44
residues of the hAd2 fiber coupled to a trimerizing
foldon domain from T-4 bacteriophage fibritin [28]
(Fig. 5A). The foldon domain has been shown to
induce trimerization in adenovirus fiber protein con-
structs [29] and was used in the minifiber to mimic the
proper oligomerization state of the full-length protein.
Proper trimerization of the minifiber construct was
confirmed by gel filtration and native gel (data not
shown). The minifiber was labeled with tetramethyl-
rhodamine (TMR) at C-terminal cysteine residues and
purified by size exclusion chromatography.
Changes in fluorescence anisotropy were used to
directly measure the binding of the TMR-labeled mi-
nifiber to the penton base. The initial reaction between
the minifiber and the penton base was assumed to be
reversible with
F þ PB $ P

where F is the minifiber, PB the penton base, and P* the
initial fiber–penton base complex. Owing to the con-
formational change of the penton base after fiber bind-
ing, the full binding expression can be represented as
F þ PB $ P


! P
with the formation of P being virtually irreversible. The
apparent K
d
measured here is the K
d
for the initial bind-
ing of minifiber. The fluorescence emission of the TMR
was assumed to be independent of the binding of the
minifiber to the penton base, because it is attached at
the C-terminal extremity of the foldon domain and is
remote from the penton base-binding region.
The system was particularly amenable to this tech-
nique, due to the relatively small size of the minifiber
( 24 kDa for the trimer) in conjunction with the large
size of the penton base ( 300 kDa for the pentamer).
K
d
values for minifiber binding to wild-type hAd2pb
B
A
Fig. 5. Sequence alignment and binding data for the minifiber–pen-
ton base complex. (A) Alignment of the N-terminal region of hAd2
fiber (accession number CAJ29207), hAd12 fiber (accession num-
ber CAJ29196), and minifiber. The residues known to interact with
the penton base are in yellow and the T4 fibritin foldon domain of
the minifiber is in red. Derivatization with 2-methyl-2,4-pentanediol
(TMR) was performed on the C-terminal cysteine residue of the
minifiber. (B) Left: fluorescence anisotropy measurement for wild-

type (wt) (red circles) and hAd2 ⁄ 12 (blue squares) with labeled
minifiber at 33 n
M for all measurements. Varying amounts of pen-
ton base were titrated in and plotted against anisotropy. Right:
competition assay for labeled versus unlabeled minifiber. The labe-
led minifiber at 15 n
M was preincubated with 3 lM penton base.
Unlabeled minfiber was titrated into the solution, and the anisotro-
py values were recorded. No dissociation of labeled minifiber was
noted with the concentrations of minifiber used.
C. Zubieta et al. Human adenovirus 2 ⁄ 12 penton base chimera
FEBS Journal 273 (2006) 4336–4345 ª 2006 The Authors Journal compilation ª 2006 FEBS 4341
and chimeric hAd2 ⁄ 12pb were 1.0 ± 0.3 lm and
1.85 ± 0.7 lm, respectively (Fig. 5B). During viral
assembly, concentrations of the fiber protein within the
nucleus of an infected cell are likely to be at least in
the low micromolar range, based upon the concentra-
tion of virions [30] and excess fiber protein [31] pro-
duced during adenoviral infection; thus, the K
d
values
measured are physiologically reasonable.
In order t o investigate the r ole o f the pr oposed con-
formational change that occurs upon fiber binding, we
attempted to disso ciate the bound minifiber from the
penton base . Previous studies have s hown that in vitro
removal of the fiber protein from adenovir us requires the
use of chaotropic salts and increased temperatures [32,33],
and no release of fiber protein has been noted even after
long-term storage of dilute solutions of virus [34].

To address these observations, we performed competi-
tion experiments using unlabeled minifiber. Unlabeled
minifiber was titrated into samples of TMR-labeled
minifiber bound to wild-type and chimeric penton
base. No decrease in fluorescence anisotropy was
observed even upon addition of 50-fold excess of un-
labeled minifiber over the calculated K
d
(Fig. 5B).
These data support the hypothesis that after fiber
binding, the conformational change occurring in the
penton base locks the fiber protein into place. Thus, as
noted previously, a full representation of the binding
equilibrium of the interaction is
F þ PB $ P

! P
where F is the fiber or minifiber protein, PB is the pen-
ton base, P* is the initial penton, and P is the penton
after conformational change. The stability of the com-
plex results from the effect of the crystallographically
observed cooperative conformational change in the
fiber protein-binding site. Based on the structure of
hAd2pb with an N-terminal fiber peptide, conforma-
tional changes will occur in the penton base upon
interaction of the fiber protein, essentially locking in
the fiber [20].
Studies with adenovirus 2 have shown that fiber loss
occurs at the cell surface and prior to endocytosis [19].
Although the mechanism of fiber dissociation from the

penton is not clear, the extreme stability of the penton
once formed and the relatively weak micromolar affin-
ity for initial complex formation has important impli-
cations for adenovirus infectivity. For successful
infection, adenovirus needs the fiber protein as a pri-
mary points of attachment to the host cell. Studies
with fiberless adenoviral particles demonstrated a sev-
eral thousand-fold decrease in infectivity [35]. Thus,
premature loss of the fiber would result in marked
decreases in infection. Once the virus is attached to
the cell surface, however, the fiber protein is not neces-
sary for the later steps in the viral cycle. After fiber
loss, the low micromolar affinity of the penton base
for fiber protein minimizes reattachment of the fiber
to the viral particle. The altered conformation of the
fiber-binding site in the hAd2 ⁄ 12 chimeric structure
led us to investigate the possibility of coupling
between the conformation of the hypervariable loop,
which contains the integrin-binding site, and the fiber-
binding site of the penton base. Based on the fluores-
cence anisotropy data presented here for the wild-type
and chimeric protein showing similar binding profiles
with relatively weak (1–2 lm) initial affinity of the
penton base for the fiber and virtually no release of
fiber once bound, adenovirus modulates fiber attach-
ment by a two-step fiber-binding process that is inde-
pendent of the conformation of the hypervariable
loop. The mechanism for fiber release is not coupled
to the hypervariable loop size or conformation but
may be influenced by the solvent environment of the

virion. In this structural study, the fiber-binding site
was in a ‘bound’ conformation, although no fiber was
present. The most likely explanation for this observa-
tion is that the switch from the fiber ‘bound’ to
‘unbound’ states of the penton base (or vice versa) is
not only dependent on the presence or absence of the
fiber itself but can also be triggered by solvent envi-
ronment effects. Owing to the cooperativity of this
switch [19], all penton bases will be in the same con-
formation. Triggering the switch to the unbound state
would clearly favor fiber release. Our results suggest
that this is not dependent on the hypervariable loop
directly, but could be due to other interactions with
the bound integrin or the particular solvent environ-
ment of the virion at the cell surface or in the initial
stages of endocytosis.
Experimental procedures
For baculovirus expression, the SF21 and Hi5 cell lines and
the Bac-to-Bac expression system and vectors were from
Invitrogen (Carlsbad, CA, USA). Protease inhibitors were
from Roche (Basel, Switzerland) and Ni-NTA resin was
obtained from Bio-Rad (Hercules, CA, USA). The minifiber
construct was a gift from the Laboratory of Glen Nemerow
at The Scripps Research Institute (TSRI) in La Jolla, CA,
USA. An MOS450 fluorimeter (Biologic, SA, Claix, France)
was used in the fluorescence anisotropy assays.
Protein expression and purification
cDNA encoding a 49-residue N-terminal truncation of
hAd2pb was cloned into a pFastbac vector as described
Human adenovirus 2 ⁄ 12 penton base chimera C. Zubieta et al.

4342 FEBS Journal 273 (2006) 4336–4345 ª 2006 The Authors Journal compilation ª 2006 FEBS
previously [20]. The hAd2 ⁄ 12pb chimera was constructed
by annealing complementary overhangs from two fragments
of the hAd2 gene. The forward oligomer 5¢-TATTT
TCAGGGCGCCATGGGATCCCCCTTCGATGCTCCC-3¢
and the reverse oligomer 5¢-TGGTTTCGGAGCGGCC
GCATTATCGCCCCTCC CGCCCTGTTCG G-3¢ were
used to introduce a Not1 site C-terminal to the RGD motif
of hAd2 in addition to an N-terminal Nco1 cloning site.
The forward o ligomer 5 ¢-TCCGAAACCAGCG GCCGCTT
TATCGCGTTA AAAC CGGT GATCAA ACCCC -3¢ and the
reverse oligomer 5¢-GTAGGCCTT TGAATTCCTCAAAA
AGTGCGGCTCGAT-3¢ were used to introduce a Not1 site
followed by the hAd12pb hypervariable loop sequence into
hAd2 in addition to a C-terminal EcoR1 site. The resulting
fragments were digested with Not1, Nco1orEcoR1, puri-
fied, and annealed. The Not1 site in the resulting gene
was removed using the complementary oligomers
5¢-GGCGGGAGGGGCGATAATTTTATCGCGTTAAAA
CCG-3¢ (forward) and 5¢-CGGTTTTAACGCGATAAAAT
TATCGCCCCTCCCGCC-3¢ (reverse).
Virus amplification was performed in monolayer SF21
cells, and protein expression was performed in HighFive cells
in shaker flasks (135 r.p.m., 27 °C, 3 days). For protein
expression, a multiplicity of infection of 5–10 was used. After
3 days of expression, cells were pelleted at 195 g for 5 min at
4 °C using a Jouan CR3 centrifuge with a T-40 rotor, and
stored at ) 80 °C. For protein purification, cells were resus-
pended in 25 mm Tris (pH 7.5) ⁄ 100 mm NaCl, plus protease
inhibitors (Roche) and lysed by sonication. Cell debris was

pelleted at 43 000 g for 45 min at 4 °C using an Avanti J-25
centrifuge with a JA25.50 rotor (Beckman Coulter, Fuller-
ton, CA, USA), and the supernatant collected. The protein
was precipitated with 30% ammonium sulfate and the
precipitant collected. After resuspension in 25 mm Tris
(pH 7.5) ⁄ 100 mm NaCl, the protein was dialyzed overnight
against the same buffer. The protein solution was concentra-
ted to 5mgÆmL
)1
and applied to a MONO Q column
(Pharmacia, Uppsala, Sweden) with a linear gradient of
100 mm to 1 m NaCl in 25 mm Tris (pH 7.5). The protein
eluted at approximately 220 mm NaCl. Fractions of interest
were buffer exchanged, concentrated to 5–10 mgÆmL
)1
and
stored at ) 80 °C.
The minifiber construct was cloned into a pEtM11 expres-
sion vector between the Kpn1 and Nco1 sites using a forward
oligomer 5¢-CTTTATTTTCAGGGCGCCATGAAGCGCG
CAAGACCGTCTGAA-3¢ and a reverse oligomer 5¢-AGCT
CGAATTCG GATCCGGTACCTCAGAAGGTAGACAG
CAGAACC-3¢. For derivitization with TMR, a Gly-Gly-Cys
sequence was introduced at the C-terminus using oligomers
5¢-CTGCTGTCTACCTTTGGAGGTTGCTGATCCGAA
TTCGAG-3¢ (forward) and 5¢-GCTCGAATTCGGATCAG
CAACCTCCAAAGGTAGACAGCA-3¢ (reverse). BL21
cells were transformed with the pETM11 construct and
grown until a D of 0.8 (600 nm) in Terrific Broth supple-
mented with phosphate b uffer and kanamycin at 37 °C.

The temperature was reduced to 27 °C and the culture
induced with 0.5 mm isopropyl-b-d-thiogalactopyrano-
side. Approximately 6 h postinduction, the cells were
harvested at 5800 g for 15 min using an Avanti J-25 cen-
trifuge with a JA-10 rotor. Cells were resuspended in
25 mm Tris (pH 7 .5) ⁄ 100 mm NaCl ⁄ 10 mm b-mercapto-
ethanol ⁄ 1 ·protease inhibitors. Cells were lysed by soni-
cation and cell debris pelleted at 47 000 g using an
Avanti J-25 centrifuge with a J A25.50 rotor. The super-
natant was collected and applied to a 2 mL Ni-NTA
column. The column was washed with lysis buffer con-
taining 10 mm imidazole and the protein eluted with
lysis buffer containing 200 mm imidazole. The His tag
was cleaved overnight at 4 °C using Tev protease, and
the His-tag and Tev were depleted by running the solu-
tion over the same Ni-NTA column. The protein was
concentrated and applied to an S200 size exclusion
column using 25 mm Tris (pH 7.5) ⁄ 100 mm NaCl ⁄ 10 mm
Tris(2-carboxyethyl)phosphine hydrochloride. Protein frac-
tions of interest were then concentrated and stored at
) 80 °C.
TMR labeling and binding assays
A five-fold excess of malemide-derivatized TMR was added
to a solution of purified minifiber and allowed to react for
 2 h. Excess TMR was removed by dialysis against 25 mm
Tris ⁄ 100 mm NaCl, and the protein was concentrated. The
labeled protein was then applied to an S75 column for fur-
ther purification.
A standard protocol for fluorescence anisotropy measure-
ments is as follows. Upon excitation at 546 nm, the fluores-

cence anisotropy of a 33 nm solution of labeled minifiber
was measured at an emission wavelength of 575 nm perpen-
dicular to the excitation vector. Either wild-type hAd2pb or
hAd2 ⁄ 12pb was titrated into the solution in  200–300 lm
increments. Anisotropy values were recorded for 60 points
over 60 s, and an average value was taken as a data point.
Titrations were continued until a stable anisotropy value
was obtained.
Fluorescence anisotropy, A, is defined as
A ¼ðI
V
 I
H
Þ=ðI
V
þ 2I
H
Þ
where I
V
and I
H
refer to the parallel and perpendicular
components of the polarized fluorescence emission. The
changes in anisotropy are a linear function with
A ¼ A
F
þðA
B
 A

F
Þð½Lig
B
=½Lig
tot
Þ
where A is the measured anisotropy value, A
F
is the aniso-
tropy of the free minifiber, A
B
is the anisotropy of the mini-
fiber bound to the penton base, [Lig
B
] is the concentration
of the bound ligand (minifiber), and [Lig
tot
] is the total con-
centration of the ligand. At any total concentration of lig-
and, the anisotropy depends on the total concentration of
C. Zubieta et al. Human adenovirus 2 ⁄ 12 penton base chimera
FEBS Journal 273 (2006) 4336–4345 ª 2006 The Authors Journal compilation ª 2006 FEBS 4343
penton base and the dissociation equilibrium constant for
the complex, K
d
, as shown by
A ¼ A
F
þ DAðfa ða
2

ð4½PB
tot
½Lig
tot
ÞÞ
1=2
g=2½Lig
tot
Þ
where a ¼ ([PB]
tot
+ [Lig]
tot
+ K
d
), DA ¼ (A
B
) A
F
), and
[PB] is the concentration of the penton base. Fitting the
anisotropy data to the previous equation gives a value for the
K
d.
Binding curves were generated in kaleidagraph and fit-
ted to the above equation. All measurements were done in
triplicate with at least eight data points per experiment.
Crystallization
Crystals were grown in hanging drops against 50%
MPD ⁄ 0.2 m ammonium phosphate⁄ 0.1 m Tris (pH 7.5) at

4 °C. A 1 lL drop of the protein solution at a concentra-
tion of 5–10 mgÆmL
)1
was added to 1 lL of well solution.
Small crystals grew within 2–3 months, reaching dimensions
of 50 · 50 · 50 lm. Microseeding of equilibrated drops
yielded bigger crystals of dimensions 100 · 100 · 100 lm
after a few weeks. Because the high MPD concentration
acted as a cryoprotectant, crystals were taken directly from
the drop and flash frozen prior to data collection.
Data collection and structure determination
Crystallographic data for hAd2 ⁄ 12pb were collected on the
microfocus ESRF beamline ID13 with the EMBL microdif-
fractometer (Maatel, Voreppe, France) [36]. Owing to the
large unit cell, an oscillation range of 0.5° was used during
data collection. Radiation damage was readily apparent
after a few exposures, which limited the amount of usable
data per exposed position to 2° (four 0.5° frames). Owing
to the small beam size used (10 lm), the crystal was reposi-
tioned after each 2°, permitting data collection from
an undamaged volume of crystal. Two large crystals
(100 · 100 · 100 lm
3
) provided 104 frames (52°) and 128
frames (64°) of usable data to a maximum resolution of
 3.5 A
˚
. A direct beam was taken in order to have a highly
precise value for the beam center. The beam center was
found and refined in mosflm [37]. All data were integrated

and scaled with xds.
molrep [38] was used to find a molecular replacement
solution with the hAd2 pentamer as a search model (pdb
code 1X9P). Based on the unit cell dimensions and Mat-
thews coefficient, three pentamers were expected per asym-
metric unit, with the full dodecahedral particle formed from
the space group symmetry operators. A clear solution with
three pentamers was found and used to generate initial
maps. Subsequent 15-fold NCS averaging and gradual
phase extension from 15 A
˚
to 3.6 A
˚
with rave [39] allowed
the generation of high-quality electron density maps. Model
building was performed in o [40] and refinements carried
out with refmac. Initially, strict NCS restraints were
applied to the model; however, in the later stages of refine-
ment, tight NCS constraints were only applied to the back-
bone, and the side chains were refined using medium
restraints. Unaveraged maps were examined to locate devia-
tions between monomers.
According to procheck [41] (Table 1), only three (Thr66,
Glu172, and His237) residues were in disallowed regions of
the Ramachandran plot. These generally lie in poorly
ordered loop regions. Residues 507–508 (C-terminus) and
residues 297–317 of the hypervariable loop were disordered
and not modeled. All figures were generated using pymol
[42]. Coordinates and structure factors have been deposited
in the RCSB Protein Data Bank under accession code 2C6S.

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