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 trimer
Trang 1adenovirus 2/12 penton base chimera
Chloe Zubieta1,*, Laurent Blanchoin2 and Stephen Cusack1
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 T¼ 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: czubieta@slac.stanford.edu
*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
Kd 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.
Trang 2and 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 aVb3, aVb5, or aVb1
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 aVb5integrin [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
Trang 3(regular arrangements of 12 pentamers), and the
chi-mera formed dodecahedral particles under
crystalliza-tion condicrystalliza-tions These crystallizacrystalliza-tion condicrystalliza-tions 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-recap-sidue 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.
Trang 4Overall structure
The hAd2⁄ 12pb construct crystallized in space group
I222with unit cell dimensions a¼ 266.50, b ¼ 292.92,
and c¼ 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
b ¼ 292.92
c ¼ 307.30
Contents of asymmetric unit 3 pentamers
Observations
R factora,b 0.13 (0.64)
Average measurement redundancy 2.9
Refinement statistics
NCS averaging statistics c
Model refinement Rcryst⁄ R free (%) e 27.5 ⁄ 32.8
Model geometry
Ramachandran plot
a
Values in parentheses refer to the highest resolution shell.
b Rfactor¼ 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.dCorrelation between the densities of all noncrystallographic
symmetry averaging (NCS)-related points Rfac¼ S|F obs ) F map | ⁄
SF obs , where Fmapis the Fourier coefficient of the back-transformed
averaged map.eTight NCS restraints were used for all backbone
atoms and relaxed NCS restraints were used for side chains during
final refinements Rcryst¼ 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.
Trang 5containing 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 flexihypervaria-ble, 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 mimickfavor-ing 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.
Trang 6protein-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
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 trimerizaoligomeriza-tion 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
Fþ 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
Fþ PB $ P! P with the formation of P being virtually irreversible The apparent Kdmeasured here is the Kdfor 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)
Kd 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 minifinum-ber 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 l M 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.
Trang 7and 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 Kd values
measured are physiologically reasonable
In order to investigate the role of the proposed
con-formational change that occurs upon fiber binding, we
attempted to dissociate the bound minifiber from the
penton base Previous studies have shown that in vitro
removal of the fiber protein from adenovirus 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 Kd (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
Fþ PB $ P! P where F is the fiber or minifiber protein, PB is the
pen-ton base, P* is the initial penpen-ton, and P is the penpen-ton
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
Trang 8previously [20] The hAd2⁄ 12pb chimera was constructed
by annealing complementary overhangs from two fragments
of the hAd2 gene The forward oligomer 5ằ-TATTT
TCAGGGCGCCATGGGATCCCCCTTCGATGCTCCC-3ằ
and the reverse oligomer 5ằ-TGGTTTCGGAGCGGCC
GCATTATCGCCCCTCCCGCCCTGTTCGG-3ằ 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 oligomer 5ằ-TCCGAAACCAGCGGCCGCTT
TATCGCGTTAAAACCGGTGATCAAACCCC-3ằ and the
reverse oligomer 5ằ-GTAGGCCTT TGAATTCCTCAAAA
AGTGCGGCTCGAT-3ằ 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, Nco1 or EcoR1,
puri-fied, and annealed The Not1 site in the resulting gene
was removed using the complementary oligomers
5ằ-GGCGGGAGGGGCGATAATTTTATCGCGTTAAAA
CCG-3ằ (forward) and 5ằ-CGGTTTTAACGCGATAAAAT
TATCGCCCCTCCCGCC-3ằ (reverse)
Virus amplification was performed in monolayer SF21
cells, and protein expression was performed in HighFive cells
in shaker flasks (135 r.p.m., 27C, 3 days) For protein
expression, a multiplicity of infection of 5Ờ10 was used After
3 days of expression, cells were pelleted at 195 g for 5 min at
4C 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 4C 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 5 mgẳmL)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 5Ờ10 mgẳmL)1and
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 5ằ-CTTTATTTTCAGGGCGCCATGAAGCGCG
CAAGACCGTCTGAA-3ằ and a reverse oligomer 5ằ-AGCT
CGAATTCG GATCCGGTACCTCAGAAGGTAGACAG
CAGAACC-3ằ For derivitization with TMR, a Gly-Gly-Cys
sequence was introduced at the C-terminus using oligomers
5ằ-CTGCTGTCTACCTTTGGAGGTTGCTGATCCGAA
TTCGAG-3ằ (forward) and 5ằ-GCTCGAATTCGGATCAG
CAACCTCCAAAGGTAGACAGCA-3ằ (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 buffer and kanamycin at 37C
The temperature was reduced to 27C 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 ởprotease inhibitors Cells were lysed by soni-cation and cell debris pelleted at 47 000 g using an Avanti J-25 centrifuge with a JA25.50 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 4C 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 200Ờ300 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
AỬ đIV IHỡ=đIVợ 2IHỡ where IV and IH refer to the parallel and perpendicular components of the polarized fluorescence emission The changes in anisotropy are a linear function with
AỬ AFợ đAB AFỡđơLigB=ơLigtotỡ where A is the measured anisotropy value, AFis the aniso-tropy of the free minifiber, ABis the anisotropy of the mini-fiber bound to the penton base, [LigB] is the concentration
of the bound ligand (minifiber), and [Ligtot] is the total con-centration of the ligand At any total concon-centration of lig-and, the anisotropy depends on the total concentration of
Trang 9penton base and the dissociation equilibrium constant for
the complex, Kd, as shown by
A¼ AFþ DAðfa ða2 ð4½PBtot½LigtotÞÞ1=2g=2½LigtotÞ
where a¼ ([PB]tot+ [Lig]tot+ Kd), DA¼ (AB) AF), and
[PB] is the concentration of the penton base Fitting the
anisotropy data to the previous equation gives a value for the
Kd.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
4C 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 lm3) 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
References
1 Hierholzer JC (1992) Adenoviruses in the immunocom-promised host Clin Microbiol Rev 5, 262–274
2 Kojaoghlanian T, Flomenberg P & Horwitz MS (2003) The impact of adenovirus infection on the immunocom-promised host Rev Med Virol 13, 155–171
3 McConnell MJ & Imperiale MJ (2004) Biology of adenovirus and its use as a vector for gene therapy Hum Gene Ther 15, 1022–1033
4 Danthinne X & Imperiale MJ (2000) Production of first generation adenovirus vectors: a review Gene Ther 7, 1707–1714
5 Amalfitano A & Parks RJ (2002) Separating fact from fiction: assessing the potential of modified adenovirus vectors for use in human gene therapy Curr Gene Ther
2, 111–133
6 Furcinitti PS, van Oostrum J & Burnett RM (1989) Adenovirus polypeptide IX revealed as capsid cement
by difference images from electron microscopy and crys-tallography EMBO J 8, 3563–3570
7 Stewart PL, Burnett RM, Cyrklaff M & Fuller SD (1991) Image reconstruction reveals the complex molecular organization of adenovirus Cell 67, 145–154
8 Stewart PL, Fuller SD & Burnett RM (1993) Difference imaging of adenovirus: bridging the resolution gap between X-ray crystallography and electron microscopy EMBO J 12, 2589–2599
9 Stewart PL & Burnett RM (1995) Adenovirus structure
by X-ray crystallography and electron microscopy Curr Top Microbiol Immunol 199, 25–38
10 Fabry CM, Rosa-Calatrava M, Conway JF, Zubieta C, Cusack S, Ruigrok RW & Schoehn G (2005) A quasi-atomic model of human adenovirus type 5 capsid EMBO J 24, 1645–1654
11 Saban SD, Nepomuceno RR, Gritton LD, Nemerow
GR & Stewart PL (2005) CryoEM structure at 9A reso-lution of an adenovirus vector targeted to hematopoietic cells J Mol Biol 349, 526–537
12 Cohen CJ, Shieh JT, Pickles RJ, Okegawa T, Hsieh JT
& Bergelson JM (2001) The coxsackievirus and
Trang 10adeno-virus receptor is a transmembrane component of the tight
junction Proc Natl Acad Sci USA 98, 15191–15196
13 Wickham TJ, Mathias P, Cheresh DA & Nemerow GR
(1993) Integrins alpha v beta 3 and alpha v beta 5
pro-mote adenovirus internalization but not virus
attach-ment Cell 73, 309–319
14 Li E, Stupack D, Bokoch GM & Nemerow GR (1998)
Adenovirus endocytosis requires actin cytoskeleton
reor-ganization mediated by Rho family GTPases J Virol
72, 8806–8812
15 Li E, Brown SL, Stupack DG, Puente XS, Cheresh DA
& Nemerow GR (2001) Integrin alpha(v)beta1 is an
adenovirus coreceptor J Virol 75, 5405–5409
16 Chiu CY, Mathias P, Nemerow GR & Stewart PL
(1999) Structure of adenovirus complexed with its
inter-nalization receptor, alphavbeta5 integrin J Virol 73,
6759–6768
17 Greber UF, Willetts M, Webster P & Helenius A (1993)
Stepwise dismantling of adenovirus 2 during entry into
cells Cell 75, 477–486
18 Medina-Kauwe LK (2003) Endocytosis of adenovirus
and adenovirus capsid proteins Adv Drug Deliv Rev 55,
1485–1496
19 Nakano MY, Boucke K, Suomalainen M, Stidwill RP
& Greber UF (2000) The first step of adenovirus type 2
disassembly occurs at the cell surface, independently of
endocytosis and escape to the cytosol J Virol 74, 7085–
7095
20 Zubieta C, Schoehn G, Chroboczek J & Cusack S
(2005) The structure of the human adenovirus 2 penton
Mol Cell 17, 121–135
21 Stewart PL, Chiu CY, Huang S, Muir T, Zhao Y, Chait
B, Mathias P & Nemerow GR (1997) Cryo-EM
visuali-zation of an exposed RGD epitope on adenovirus that
escapes antibody neutralization EMBO J 16, 1189–1198
22 Fuschiotti P, Schoehn G, Fender P, Fabry CM, Hewat
EA, Chroboczek J, Ruigrok RW & Conway JF (2006)
Structure of the dodecahedral penton particle from
human adenovirus type 3 J Mol Biol 356, 510–520
23 Chen XS, Garcea RL, Goldberg I, Casini G & Harrison
SC (2000) Structure of small virus-like particles
assembled from the L1 protein of human papillomavirus
16 Mol Cell 5, 557–567
24 Chroboczek J, Gout E, Favier AL & Galinier R (2003)
Novel partner proteins of adenovirus penton Curr Top
Microbiol Immunol 272, 37–55
25 Galinier R, Gout E, Lortat-Jacob H, Wood J &
Chro-boczek J (2002) Adenovirus protein involved in virus
internalization recruits ubiquitin-protein ligases
Biochemistry 41, 14299–14305
26 Chelvanayagam G, Heringa J & Argos P (1992)
Anat-omy and evolution of proteins displaying the viral
cap-sid jellyroll topology J Mol Biol 228, 220–242
27 Anand K, Pal D & Hilgenfeld R (2002) An overview on
2-methyl-2,4-pentanediol in crystallization and in
crys-tals of biological macromolecules Acta Crystallogr D Biol Crystallogr 58, 1722–1728
28 Guthe S, Kapinos L, Moglich A, Meier S, Grzesiek S & Kiefhaber T (2004) Very fast folding and association of
a trimerization domain from bacteriophage T4 fibritin
J Mol Biol 337, 905–915
29 Papanikolopoulou K, Forge V, Goeltz P & Mitraki A (2004) Formation of highly stable chimeric trimers by fusion of an adenovirus fiber shaft fragment with the foldon domain of bacteriophage t4 fibritin J Biol Chem
279, 8991–8998
30 Green M & Pina M (1963) Biochemical studies on ade-novirus multiplication IV Isolation, purification, and chemical analysis of adenovirus Virology 20, 199–207
31 Pettersson U, Philipson L & Hoglund S (1968) Struc-tural proteins of adenoviruses II Purification and char-acterization of the adenovirus type 2 fiber antigen Virology 35, 204–215
32 Boudin ML, Moncany M, D’Halluin JC & Boulanger PA (1979) Isolation and characterization of adenovirus type
2 vertex capsomer (penton base) Virology 92, 125–138
33 Pettersson U & Hoglund S (1969) Sructural proteins of adenoviruses 3 Purification and characterization of the adenovirus type 2 penton antigen Virology 39, 90–106
34 Boudin ML & Boulanger P (1981) Antibody-triggered dissociation of adenovirus penton capsomer Virology
113, 781–786
35 Von Seggern DJ, Chiu CY, Fleck SK, Stewart PL & Nemerow GR (1999) A helper-independent adenovirus vector with E1, E3, and fiber deleted: structure and infectivity of fiberless particles J Virol 73, 1601–1608
36 Perrakis A, Cipriani F, Castagna JC, Claustre L, Burg-hammer M, Riekel C & Cusack S (1999) Protein micro-crystals and the design of a microdiffractometer: current experience and plans at EMBL and ESRF⁄ ID13 Acta Crystallogr D Biol Crystallogr 55, 1765–1770
37 Leslie AGW (1992) Recent changes to the MOSFLM package for processing film and image plate data Joint CCP4 + ESF-EAMCB Newsl Protein Crystallogr 26
38 Vagin A & Teplyakov A (2000) An approach to multi-copy search in molecular replacement Acta Crystallogr
D Biol Crystallogr 56, 1622–1624
39 Kleywegt GJ & Jones TA (1999) Software for handling macromolecular envelopes Acta Crystallogr D Biol Crystallogr 55, 941–944
40 Jones TA, Zou JY, Cowan SW & Kjeldgaard M (1991) Improved methods for building protein models in elec-tron density maps and the location of errors in these models Acta Crystallogr A 47, 110–119
41 Laskowski RA, MacArthur MW, Moss DS & Thornton
JM (1993) PROCHECK: a program to check the stereo-chemical quality of protein structures J Appl Crystal-logr 26, 283–291
42 DeLano WL (2002) The PyMOL Molecular Graphics System.DeLano Scientific, San Carlos, CA