Solution properties of full-length integrin
a
IIb
b
3
refined
models suggest environment-dependent induction of
alternative bent
⁄
extended resting states
Camillo Rosano
1
and Mattia Rocco
2
1 Nanobiotecnologie, Istituto Nazionale per la Ricerca sul Cancro (IST), Genova, Italy
2 Biopolimeri e Proteomica, IST, Genova, Italy
Introduction
Integrins are heterodimeric transmembrane (TM) cellu-
lar receptors involved in mechanical anchoring and
two-way signaling [1]. Each a and b subunit has a
modular structure with a large extracellular portion,
a single TM region and a cytoplasmic domain [1–3].
The integrin activation mechanism is regulated by
conformational changes, the details of which have not
yet been fully elucidated [2,3]. X-ray crystallography
Keywords
blood coagulation; hydrodynamics;
modeling; modular proteins; protein
structure
Correspondence
M. Rocco, Biopolimeri e Proteomica, IST

a
IIb
b
3
supports the bent ⁄ closed resting form. However, only an extended ⁄ -
closed model matches well the hydrodynamics of either octyl-glucoside-sol-
ubilized or nanodiscs-embedded resting a
IIb
b
3
, suggesting that different
solubilization strategies and substrate interactions might operate a confor-
mational selection between alternative, stable states. Furthermore, extende-
d ⁄ open models are required to match the electron tomography map and
the hydrodynamics following the priming-induced b
3
hybrid domain swing-
out, but without immediate full tail separation. Importantly, both extension
and opening transitions can occur by pivoting at the recently identified b
3
hinge point, which does not appear to be freely flexible. The structure and
mechanism of action of integrins thus seem to depend on discrete transi-
tions and to be more tightly coupled to the local environment than previ-
ously thought.
Abbreviations
bc, bent ⁄ closed; DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DMPG, 1,2-dimyristoyl-sn-glycero-3-phospho-(1¢-rac-glycerol); DPPC,
1,2-dipalmitoyl-sn-glycero-3-phosphocholine; ec, extended ⁄ closed; EGF, epidermal growth factor; EM, electron microscopy; eotc,
extended ⁄ open ⁄ tails crossed; eots, extended ⁄ open ⁄ tail separated; ET, electron tomography; NMA, Normal Modes Analysis; OG, octyl-
glucoside; pec, partially extended ⁄ closed; PSI, plexin ⁄ semaphorin ⁄ integrin; SANS, small-angle neutron scattering; TEM, transmission
electron microscopy; TM, transmembrane.

3
hybrid domain [6]) taking place
without the requirement of full separation of the TM
helices. Since then, crystallographic structures of the
a
IIb
b
3
, a
v
b
3
and a
x
b
2
ectodomains [11–13] and two
NMR-based structures of the a
IIb
b
3
TM helices – one
embedded in a small bicelle [14] and the other solubi-
lized in a mixed solvent and including the cytoplasmic
domains [15] – have been published. Furthermore, new
low-resolution data of full-length a
IIb
b
3
have very

NMR-based structures of the TM helices ⁄ cytoplasmic
domains for the resting integrin state, while for the
primed state we employed the computer models [18,19]
utilized in our previous work [10]. Particular care was
exerted when modeling the major, still-unresolved,
loop in the calf-2 module of a
IIb
, and we resorted to
using ab initio modeling procedures. Extended ⁄ closed
and extended ⁄ open models were derived from the fully
bent ⁄ closed crystallographic model by fitting to the ET
map, and two series of intermediate models were
obtained by morphing between these initial and final
conformations. The models were then assessed by com-
paring their hydrodynamic and conformational param-
eters (computed using the new UltraScan SOlution
MOdeler (US-SOMO) bead-modeling implementation
[20–22]) with experimental data [10,16,17]. While the
fully bent ⁄ closed crystallographic model was incompat-
ible with all the available solution data for resting
a
IIb
b
3
, differences remained between a partially exten-
ded ⁄ closed SANS-complying model and the extended ⁄ -
closed ET ⁄ hydrodynamics-based model. However, the
SANS data could also be interpreted as deriving from
a mixture of bent and extended conformations. More-
over, only an extended ⁄ open model without full tail

(E764-D774) was modeled using
ModLoop [23], whereas the G75-S78 and D477-Q482
loops in b
3
were taken from the new a
v
b
3
ectodomain
structure, 3IJE [12]. Given their probable flexible nat-
ure, no attempt was made to fully optimize the confor-
mation of these segments. As for the still-unresolved
long loop at the end of the a
IIb
calf-2 module (G840-
Q873), alignment of all human integrin a subunits
using ClustalW [24] showed that those with a proven
or putative cleavage site in the calf-2 module have
longer loops between conserved cysteine residues than
uncleaved integrins (Supporting information Fig. S1).
Two structured stretches (mainly a-helical, some
extended) were consistently predicted by ab initio mod-
eling using Robetta [25] within this region, mostly
preserved by the cleavage sites (see Fig. S1; a gallery
of predicted structures is shown in Fig. S2). While a
C. Rosano and M. Rocco Refined a
IIb
b
3
models

subunit in the ET map [9]. The
a
IIb
b-propeller and the b
3
bA ⁄ hybrid ⁄ plexin ⁄ sema-
phorin ⁄ integrin (PSI) domains of the 3FCS structure
[11] were inserted as a single block to preserve the
a
IIb
⁄ b
3
interface. The b
3
I-EGF1-4 and bTD domains
were then added in the bent conformation, superim-
posing the latter on its counterpart taken from the
A2b-5 ⁄ ET original model. While a span of $ 20° was
very recently found for the interdomain angle between
AB
CDE
FG
HIJ
Fig. 1. An overview of the new, refined models. Models A2bB3–bc (panels A and F), A2bB3-pec (panels B and G), A2bB3-ec (panels C
and H), A2bB3-eotc (panels D and I) and A2bB3-eots (panels E and J), are shown as ribbons (protein only, panels A–E) and as surface
(protein) and space-filling (carbohydrates and OG moieties) representations (panels F–J). The a
IIb
b
3
modules are indicated in panel E, and

with the bA ⁄ hybrid ⁄ PSI head domain, we first gener-
ated a pathway of 12 structures from the fully closed
to the complete swung-out hybrid domain (taken from
the 3FCU structure [6]), using the Yale morph server
[27]. Aligning all these structures on the bA domain in
the A2bB3-extended ⁄ closed (ec) model framework
revealed a conformation that could be linked to the
extended I-EGF1-4 ⁄ bTD segment. A swing-out, of
$ 6°, of the hybrid domain was present in this confor-
mation, consistent with that of $ 10° observed in the
a
v
b
3
ectodomain fitted in the TEM map [7]. Finally,
after introduction of the mature a
IIb
cut at R856 [28],
Robetta [25] was again employed to remodel the
G847-R856 and D857-C879 stretches.
While an initial version of this work was undergoing
evaluation, a SANS study of native, full-length a
IIb
b
3
solubilized in Triton X-100 was published [16]. In that
study, low-resolution ($ 20 A
˚
) shape reconstruction
from the I(q) versus q profiles using dammin [29] and

could also derive from a mixture of at least two well-
defined conformations. This was confirmed by the
green curve in Fig. 2, which was obtained by averag-
ing, in a 1 : 1 ratio, the p(r) versus r of models
A2bB3-bc and A2bB3-ec. Although we did not
attempt any further refinement, it is conceivable that a
closer match could be obtained by mixing differently
bent and extended models in appropriate ratios.
Finally, to allow a comparison to be made with the
hydrodynamics of the other models, the OG moieties
were re-attached to the A2bB3-pec model.
The transition to the extended-open forms was then
achieved, starting from the A2bB3-ec model, by first
restoring to 143° the angle between the thigh and calf-
1 domains, and then superimposing the a
IIb
b-propeller
and the b
3
bA ⁄ hybrid ⁄ PSI domains of the 3FCU
structure [6]. An initial extended ⁄ open ⁄ tail separated
model [A2bB3-extended ⁄ open ⁄ tail separated (eots);
Fig. 1E,J] was then completed by allowing the b
3
I-EGF1-4 and bTD domains to follow the swing-out,
and repositioning the b
3
TM helix on the same plane
of its a
IIb

segments and re-attaching
the NMR-based cytoplasmic domains. The b
3
subunit
was then reconnected by first pivoting back the
I-EGF3-4 and bTD domains by $ 29° along the C473-
C503 hinge, and then performing a slight rotation of
the bTD module at the main chain of D606 to dock it
against the calf-2 domain with its C-terminus in prox-
imity of the N-terminus of the b
3
‘open’ TM helix.
Overall evaluation of the models
The five new a
IIb
b
3
models are shown in ribbon repre-
sentation in Fig. 1A–E, with their color-coded mod-
ules indicated in Fig. 1E. Surface and space-filling
representations are then proposed in Fig. 1F–J, where
the ET map [9] is superimposed to the fully extended
models. Note how the a
IIb
extracellular modules, and
the b
3
bA module in the extended ⁄ closed conforma-
tion, fit extremely well in the map, with the b
3

ð20;wÞ
>
w
and
<D
0
tð20;wÞ
>
z
) for resting and primed a
IIb
b
3
[10]. As seen,
the fully bent and partially extended models of the
resting integrin are incompatible with the experimental
data, while the extended ⁄ closed model is in excellent
agreement, as previously noted for the less-refined
A2b-5 ⁄ ET model [10]. As for the extended ⁄ open struc-
tures, maintaining the TM helices in contact leads to
hydrodynamic values in much better agreement (well
within the experimental range) than those of the full
swing-out model.
While this work was being prepared for resubmis-
sion, another work concerning the a
IIb
b
3
structure
appeared [17]. In this study, purified full-length a

used experimentally [17]. The His-tag and TEV prote-
ase sequence (MGHHHHHHDYDIPTTENLYFQG),
which was absent in the atomic model but present in
the original MSP1D1 construct and not removed
by Ye et al. [17], was modeled by a combination of
ab initio modeling using Robetta [25] and insertion
from the 3GZH.pdb structure (YDIPTTENLYFQG).
It was then grafted at the two N-termini of the
MSP1D1-DMPC ⁄ DMPG nanodisc structure, again
without any further optimization. The computed
molecular mass for this nanodisc model,
156 853 gÆmol
)1
, is in good agreement with the experi-
mental value, of 181 500, measured by Ye et al. [17],
which was recalculated after correcting for the com-
puted

v of the nanodisc, 0.875 cm
3
Æg
)1
(F. Ye and
A. Bobkov, personal communication). No further opti-
mization of the DMPC : DMPG ratio was attempted,
as it would have only slightly affected the computed
hydrodynamic parameters. About 18 DMPC : DMPG
molecules were then removed to make space for the
TM helices of the integrins, either from the center of
the nanodisc or close to the protein belt, to take into

C. Rosano and M. Rocco Refined a
IIb
b
3
models
FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS 3195
that the fully bent integrin-conformation is at odds
(+12 ) 14%) with the solution data, whereas the
extended ⁄ closed conformation is in very good agree-
ment with them (+1 ) 3%).
Discussion
The revised a
IIb
b
3
models, which are now based on
more complete structural evidence, allow a better eval-
uation of proposed alternative conformational states
and the supposed transitions between them. To begin
with, the fully bent, crystallographic state observed for
the a
v
b
3
, a
IIb
b
3
and a
x

and should clearly engulf also the cytoplasmic
domains. This could potentially deregulate any confor-
mational control exerted at the inner membrane inter-
face, which should instead be preserved in the
OG- and nanodiscs-solubilized samples.
The new a
IIb
b
3
and a
x
b
2
ectodomain structures have
been used to reinforce the scheme calling for bent rest-
ing integrins with a transition to fully extended, open,
tails separated structures following activation [11,13].
The recently published negative-staining EM images,
derived either from ectodomain constructs [13] or from
full-length, nanodisc-solubilized samples [17], also
show a predominance of bent forms in the resting
state. Regarding the ectodomain studies, an important
question can be raised of whether it is more ‘physio-
logical’, for example, a truncated construct in which
the TM and cytoplasmic regions are absent, immobi-
lized either in a crystal lattice or on an EM grid, or is
a full-length, native molecule isolated in a small
micelles-forming mild detergent or in a confined lipid
bilayer. While good arguments could be made either
way, is interesting to note that previous TEM work [7]

3
and the refined
models.
Experimental ⁄ models <s
0
ð20;wÞ
>
w
(S)
Percentage ± SEM or
percentage difference <D
0
tð20;wÞ
>
z
(F)
Percentage ± SEM or
percentage difference
a
IIb
b
3
resting 8.18 ± 0.07
a
± 0.9 3.07 ± 0.08
a
± 2.6
A2bB3-bc (bent ⁄ closed) 9.08 + 11.0 3.31 + 7.8
A2bB3-pec (partially extended ⁄ closed) 8.99 + 9.9 3.27 + 6.5
A2bB3-ec (extended ⁄ closed) 8.20 + 0.2 2.98 )2.9

3
models C. Rosano and M. Rocco
3196 FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS
phospholipid moieties are confined within the scaffolds
of the nanodiscs. Furthermore, the extended ⁄ closed
state appears to be a stable conformation in purified,
solubilized full-length a
IIb
b
3
, which is fully able to
make the transition to the extended ⁄ open state upon
priming and to revert back to the extended ⁄ closed
state when the priming agent is removed [35]. In addi-
tion, the transition to the open state can take place on
the cell membrane with changes in the integrin height
that vary widely from system to system (e.g. [36]; see
also [37,38], and references therein). Instead, the recent
studies with nanodiscs-embedded full-length a
IIb
b
3
[17]
provide apparently contradictory results: while the
hydrodynamic data are fully consistent with an
extended resting integrin, the EM images show a pre-
dominance of bent forms. Importantly, the shape dis-
tribution appears to be rather bimodal, with the
molecules assuming either a bent or an extended form,
and an absence of well-defined intermediate conforma-

MSP1D1 identical subunits are orange and
red, respectively. The carbohydrates are
shown as gold sticks, and the DMPC and
DMPG lipids are shown in space-filling
mode (light grey, DMPC; slate grey,
DMPG).
C. Rosano and M. Rocco Refined a
IIb
b
3
models
FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS 3197
observed in the EM images of the a
x
b
2
ectodomain
[13]. Thus, true to the previously proposed ‘switch-
blade’ extension mechanism [39], the conformational
change indeed appears to be snap-like, albeit not
directly linked to priming or activation. In this light, it
could be hypothesized that interactions with the EM
grid favor a snap-back to the bent conformation, thus
offering an explanation for the apparent contradiction
between the EM and the hydrodynamic data [17].
Interestingly, the recent a
IIb
b
3
-nanodiscs EM study

as well act as a ‘universal’ joint, coupling the swing-
out to a simple conformational change in the TM heli-
ces. In this respect, recent work has claimed that the
b
3
S527F mutation in a
IIb
b
3
induces the high-affinity
state by hindering the adoption of the bent conforma-
tion [41]. However, the closest residue in the ‘head’
region, S401 of a
IIb
, is more than 20 A
˚
away in the
bent conformation, while the S527F mutation perturbs
a cluster of polar residues (R671 and N675 in a
IIb
,
R498, D524, S527, R530, D546 and Y556 in b
3
) both
in the bent and in the extended-closed conformations.
Furthermore, the S527-containing C523-C544 loop
appears to be only slightly perturbed by the pivoting
movement. Therefore, it seems more likely that this
mutation affects only the swing-out, thus favoring the
high-affinity state, without any implication for the

from several antibody-binding studies (see [3], and
references therein), recently revisited on the basis of
the new a
x
b
2
structure [13]. We have mapped the
Table 2. Comparison between experimental and computed sedi-
mentation coefficients for full-length a
IIb
b
3
embedded in the
MSP1D1-DMPC ⁄ DMPG nanodiscs and the new models.
Experimental ⁄ models <s
ð20;wÞ
>
w
(S)
Percentage
difference
a
IIb
b
3
resting 9.02
a
na
A2bB3-bc-ndc (bent ⁄ closed,
nanodisc, centered)

3
models C. Rosano and M. Rocco
3198 FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS
antibody-binding sites identified in a
L
b
2
as markers of
activation [43–45] on our refined models A2bB3-bc,
A2bB3-ec and A2bB3-eotc (see Supporting informa-
tion Fig. S3). While the patches corresponding to the
NKI-L16 and AO3 anti-a
L
antibody-binding sites seem
to be more exposed in the extended ⁄ closed and exten-
ded ⁄ open conformations (Fig. S3, panels B–C), they
do not appear to be buried in the bent ⁄ closed confor-
mation (Fig. S3, panel A). Noting that the majority of
these epitope residues in the thigh module are close to
the junction with the b-propeller module, it is possible
that the differences in antibody binding stem from
changes in the relative orientation of these two mod-
ules. Indeed, a variation of $ 18° is observed between
the thigh ⁄ b-propeller modules in 10 independent mole-
cules in a
x
b
2
crystals [13], suggesting a degree of flexi-
bility similar to that present at the I-EGF1 ⁄ 2 interface.

tion of the lower b
3
leg already accompanies extension,
bringing the activating residues into contact with other
residues in the a
IIb
subunit. In addition, some caution
should be exerted in transferring the results of anti-
body-binding and mutational studies, carried out on
different integrins, to the a
IIb
b
3
integrin, which might
be controlled by a different conformational regulatory
system. Given the complexity of the integrin activa-
tion ⁄ signaling network (e.g. see [46] and references
therein) further experimental work is clearly needed to
fully resolve these issues.
The putative structured segment in the calf-2 loop
that was consistently generated by ab initio modeling
in all integrin a subunits that undergo post-transla-
tional cleavage could be physiologically relevant, but
caution should be exerted to avoid overinterpretations
being made. A more extensive study, for instance
involving repeating the ab initio generation after
scrambling the loop sequences, should be performed to
strengthen the prediction, but this is outside the scope
of the present study. In any case, that this loop was
not resolved in any ectodomain crystal structure indi-

wise. Although the thermodynamic cost of such a huge
conformational change could still be manageable, its
physiological need is unclear, and alternative explana-
tions for the role of the bent conformation, such as the
‘packaging for transport’ hypothesis that we have
already proposed [10], should be investigated on a case-
by-case basis. The refined models presented here, which
have been deposited in the public Protein Model Data-
Base (PMDB; should also
aid the design of mutational studies aimed to fully clar-
ify these important issues. While we recognize that they
clearly cannot provide definitive atomic details of unre-
solved, modeled segments, such as the calf-2 long loop
and the relative orientation of the ectodomain ⁄ TM
regions, or of the changes induced by extension not seen
in crystal studies, we exerted great care and chose con-
servative assumptions during modeling. Our refined
models should also allow more realistic steered mole-
cular dynamics simulations of the conformational
C. Rosano and M. Rocco Refined a
IIb
b
3
models
FEBS Journal 277 (2010) 3190–3202 ª 2010 The Authors Journal compilation ª 2010 FEBS 3199
transitions of integrins (e.g. [11]) to be performed, for
instance by the use of nanodiscs-embedded models.
Materials and methods
Molecular models were built and refined mostly as previ-
ously described [10]. The Modeller ModLoop utility [23]

Æg
)1
[10]. The

v values for
DMPC and DMPG were 0.973 cm
3
Æg
)1
(adjusted at 20 °C
from the experimental value at 30 °C [48]) and 0.925
cm
3
Æg
)1
(adjusted at 20 °C from the calculated [49] value at
25 °C), respectively. The calculated

v value at 20 °C for the
MSP1D1 nanodisc scaffold protein with the initial methio-
nine, the His-tag and the TEV protease site, was 0.731 cm
3
Æ
g
)1
. For the nanodiscs-embedded integrins, the calculated

v
at 20 °C thus was 0.776 cm
3

Acknowledgements
We thank R.R. Hantgan (Wake Forest University,
NC, USA) for comments. We are grateful to M.A. Ar-
naout (Harvard Medical School, Charlestown, MA,
USA) for very kindly providing us with the coordinates
of the new a
v
b
3
ectodomain structure before public
release; to F. Ye, M. Ginsberg (UCSD, CA, USA) and
A. Bobkov (The Burnham Institute, CA, USA) for pro-
viding important details of their experimental work and
feedback on our calculations of the nanodiscs proper-
ties; and to A. Shih and S.G. Sligar (University of Illi-
nois at Urbana-Champaign, IL, USA) for providing a
nanodisc atomic model and related information. We
are indebted to E. Brookes (UTHSCSA, San Antonio,
TX, USA) for his constant and timely improvements to
the US-SOMO program. This work was partially sup-
ported by the Italy-USA project ‘Farmacogenomics
oncology – Oncoproteomics’ (Grant 527B ⁄ 2A ⁄ 3) to
MR. The models accession codes in the PMDB
database are PM0076386 (A2bB3-bc), PM0076372
(A2bB3-pec), PM0076362 (A2bB3-ec), PM0076363
(A2bB3-eotc), and PM0076371 (A2bB3-eots).
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Supporting information
The following supplementary material is available:
Fig. S1. Alignment of the long loop region in the calf-
2 module of integrin’s a subunits.
Fig. S2. A gallery of structures predicted by Robetta
[20] for the long loop in the calf-2 region of integrins’
a subunits without the insertion domain.
Fig. S3. Mapping the a
L
and b
2
activation-sensitive
antibodies binding sites on the a
IIb
b3 models A2bB3-
bc (panel A), A2bB3-ec (panel B) and A2bB3-eotc
(panel C), shown in surface representation mode with
the relevant residues highlighted in space-filling mode.