641
Coming to grips with integrin binding to ligands
Opinion
M Amin Arnaout*‡, Simon L Goodman† and Jian-Ping Xiong*
Integrins are αβ heterodimeric cell-surface receptors that are vital
to the survival and function of nucleated cells. They recognize
aspartic-acid- or a glutamic-acid-based sequence motifs in
structurally diverse ligands. Integrin recognition of most ligands is
divalent cation dependent and conformationally sensitive. In
addition to this common property, there is an underlying binding
specificity between integrins and ligands for which there has
been no structural basis. The recently reported crystal structures
of the extracellular segment of an integrin in its unliganded state
and in complex with a prototypical Arg–Gly–Asp (RGD) ligand
have provided an atomic basis for cation-mediated binding of
aspartic-acid-based ligands to integrins. They also serve as a
basis for modelling other integrins in complex with larger
physiologic ligands. These models provide new insights into the
molecular basis for ligand binding specificity in integrins and its
regulation by activation-driven tertiary and quaternary changes.
Addresses
*Renal Unit, Leukocyte Biology and Inflammation Program, Structural
Biology Program, Massachusetts General Hospital, and Harvard
Medical School, 149 13th Street, Charlestown, MA 02129, USA
† Department of Biomedical Research, Oncology, Merck KGaA,
Darmstadt 64271, Germany
‡ e-mail: arnaout@receptor.mgh.harvard.edu
Current Opinion in Cell Biology 2002, 14:641–651
0955-0674/02/$ — see front matter
© 2002 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S095506740200371X
Abbreviations
ADMIDAS adjacent to MIDAS
EGF
epidermal growth factor
FB
fibrinogen
FN
fibronectin
LIMBS
ligand-associated metal-binding site
MIDAS
metal-ion-dependent-adhesion site
PSI
plexins, semaphorins and integrins
RGD
Arg–Gly–Asp (ligand)
VCAM
vascular cell adhesion molecule
Introduction
The formation, remodelling and maintenance of tissues and
organs is achieved through dynamic links between cells and
their microenvironment, across which complex chemical and
physical cues communicate back and forth. Integrins, type I
transmembrane αβ heterodimeric receptors, provide these
links in nucleated cells. On one side of the plasma membrane,
integrins recognise structurally diverse components of the
extracellular matrix (ECM) and adjacent cells as well as
plasma proteins, and on the other side they connect directly,
through their short cytoplasmic regions, to the cell’s locomotive engine, the cytoskeleton (reviewed in [1]). Integrin
binding to extracellular ligands is translated into formation
and remodelling of focal adhesions, which are responsible for
the changes in cellular shape and tissue organisation
(reviewed in [2]). Signals from the cell interior are communicated to the integrin ectodomain, instructing it to modify its
grip, when cells need to move around or reorganise their
extracellular microenvironment. A delicate balance therefore
exists between inside–out and outside–in signals transduced
across integrins.
An extensive body of literature over the past three decades
has elucidated the nature and complexity of the integrin
receptors involved, the spectrum of ligands they recognise,
and the signalling pathways involved in regulating their
functions. Totally missing was an atomic structure of
unoccupied and liganded integrins, which would enable a
better interpretation of the vast amount of accumulated
functional data and help plan future investigations on a
sound structural basis. In this article, we review the recent
advances we have made in elucidating the crystal structure of
an integrin in its unliganded and RGD-liganded states. We also
attempt to put some of the vast body of current biochemical
data on integrin–ligand interactions in a structural context.
Determinants of integrin recognition of ligands
Integrin–ligand interactions are determined by several
factors. One major determinant of ligand binding specificity
is the subunit composition of an integrin. In mammals, 18 α
and eight β subunits combine to form 24 integrins. One half
of the integrin α subunits contain an extra von Willebrand
factor A-type domain (αA or I domain;) and are known as αA
domains (reviewed in [3]). The α and β subunits are also
subject to alternative splicing [4] and post-translational
modifications [5–7], enriching this structural diversity.
A second major factor in determining integrin binding to
ligands is the presence of integrin recognition sequences in
ligands [8]. Integrins bind many ligands in a divalentcation-dependent manner (reviewed in [9]). These include
a large number of extracellular matrix proteins (e.g. various
collagens, fibronectins, vitronectin, laminins, von Willebrand
factor and thrombospondins), counter-receptors (e.g. VCAM-1,
ICAMs, and generally members of the immunoglobulin
superfamily) and plasma proteins. Numerous pathogens
and venom components also utilise integrins to initiate
infection [10–14]. Despite the structural diversity of
integrin ligands, they have in common an exposed aspartic
acid or glutamic acid residue, critical for recognition by
integrins, that is generally found within extended flexible
loops [15–18]. The degree of solvency of the ligand
aspartic acid or glutamic acid can be subject to regulation.
The ligand aspartic acid or glutamic acid is present within
defined recognition sequences found in one or more copies
within a ligand. Aspartic-acid-based sequences (e.g. RGD,
642
Cell-to-cell contact and extracellular matrix
LDV, KGD, RTD and KQAGD [single letter amino acid
code]) bind to the majority of integrins [19]. Nine
integrins, however, bind to glutamic acid- (rather than
aspartic acid-) based motifs in ligands; these characteristically
contain the αA domain, which mediates ligand binding in
these cases [20].
The third factor that affects integrin–ligand interactions is
divalent cations. The glutamic acid or aspartic acid in ligands forms a ternary complex with receptor-bound
divalent cations [21,22••,23••] in αA-containing or -lacking
integrins, respectively. While Mg2+ and Mn2+ support
physiological ligand binding at a broad range of concentrations,
Ca2+ does so in the micromolar range in both αA-containing
[24] and -lacking integrins [25]. At millimolar concentrations (those found normally in the plasma), however, Ca2+
is generally inhibitory [25–27]. It has been suggested that
millimolar concentrations of Ca2+ bind at an allosteric
Ca2+-specific binding site in the β subunit and increase
the rate of ligand dissociation [25]. In vivo, regulation of
integrins by cations may be important in remodelling
tissue, such as bone and gut mucosa, and healing wounds,
instances where local concentrations of free or ECM-bound
cations can vary [28].
The fourth important factor is the activation state of an
integrin, which plays a major role in ligand recognition.
Low-affinity interactions mediated by clustered integrins
[29–34] are probably responsible for tethering and rolling
of cells [34,35•]. These interactions are enhanced by
preclustered or multivalent ligands [36] as well as by the
cytoskeleton [37]. Firm adhesion however requires highaffinity interactions [34,35•,38•]. High-affinity binding of
integrins to physiological ligands requires a conformational
switch in the receptors (activation) from the low- to the
high-affinity state. This switch is triggered naturally by
inside–out signals, presumably acting to release intracellular
constraints exerted on the cytoplasmic regions of an
integrin [39]. High-affinity binding of pathogens or their
products, by contrast, is generally independent of the
activation state of the integrin. Integrin activation can also be
induced artificially by deleting the transmembrane/cytoplasmic segments, using certain monoclonal antibodies, by
breaking extracellular constraints, or by limited proteolysis
(reviewed in [40•]). Tertiary and quaternary rearrangements
occur extracellularly in association with this ‘activation’
switch [41–45].
Tertiary changes altering the shape and charge properties
of the ligand-binding interface have been demonstrated
directly in the isolated αA domains from various integrins
[46,47]. Biochemical studies also suggest that the RGDbinding site becomes more accessible upon receptor
activation [48]. Function-altering monoclonal antibodybinding studies suggest that quaternary changes unmask
ligand-binding sites lying near the interface between α
and β subunits [45]. Ligand binding in turn induces
neo-epitopes for monoclonal antibodies and was reported
to cause separation of the ligand-binding αβ interface of
detergent-solubilised receptors [49].
Ligand- and cation-binding sites defined
before elucidation of the integrin structure
The integrin heterodimer has a jellyfish-like appearance
by electron microscopy (EM), with a globular ‘head’ and
two ‘legs’ (reviewed in [3]). Biochemical and EM studies
have shown that the head region contains the ligandbinding site(s) [50,51]. In αA-containing integrins, ligand
binding is mediated by the αA domain [20]. This domain
belongs to the dinucleotide-binding (Rossmann) fold
[21,52], well known for undergoing tertiary-structure
changes in other proteins. A single bound divalent cation
is found at the apex of αA, coordinated by oxygencontaining sidechains from three loops, one containing an
Asp–X–Ser–X–Ser motif (where X is any amino acid), the
second an invariant threonine and the third an invariant
aspartic acid. We collectively refer to these residues as the
metal-ion-dependent adhesion site (MIDAS) motif [21].
In the unliganded (closed) conformation, shown not to
bind physiological ligands [47,53], a water molecule
provides the sixth metal coordination site. In the second
liganded (open) form, a ligand glutamic acid residue forms
a direct bond with the metal ion, replacing the water
molecule [21,46,47]. Ligand-binding specificity then arises
from adjacent residues on the MIDAS face contacting
complimentary sites in the ligand [46,53]. We have
proposed that the active and inactive states exist in an
equilibrium on the cell surface, in which the closed
conformation is favoured in resting cells. Cellular
stimulation shifts this equilibrium in favour of the open
state [47,53].
In αA-lacking integrins, ligand binding is mediated by a
predicted β-propeller domain [54,55] from the α subunit
[56–59], and a predicted αA-like domain (βA) [21] from
the β subunit [60–63]. Most studies suggested that βA
binds ligand through a putative MIDAS-like motif (by
analogy with αA) [64–68]. In several studies, ligand-binding specificity regions were mapped to surface loops at the
top of the propeller (facing the β subunit) [56,69,70] and to
an disulphide-flanked loop in βA (the ligand specificity
loop) [71]. A significant minority of studies (reviewed in
[40•]), however, mapped ligand binding at the bottom of
the propeller. Even in those studies mapping the binding
site to the propeller’s top, it remained unclear whether
αA-lacking integrins have a common ligand-binding site
[72,73], multiple distinct sites [74–78] or separate but
allosterically linked sites [79].
The MIDAS motif of αA binds Mn2+ or Mg2+ with high
(micromolar) affinity and Ca2+ with low (millimolar) affinity
[20,80]. Three additional metal-binding regions were
predicted in the extracellular segment of integrins: one
in βA [21,81], another in the EF-hand-like sequences at
the bottom of the propeller [82], and the third in the
propeller’s central core [55].
Coming to grips with integrin binding to ligands Arnaout, Goodman and Xiong
643
Figure 1
Ribbon drawing of the domain and quaternary
structure of extracellular αVβ3. The protein is
bent at a highly flexible region (the genu,
arrow) (a). When extended at the genu [22],
the structure assumes the more familiar look
revealed previously by EM, with a clearly
visible head resting on two legs (b). The 12
domains are labelled in (b). The PSI, EGF1
and EGF2 domains are disordered (grey): the
tracing shown for PSI is approximate and the
translated EGF3 and EGF4 domains have
been used to show the approximate location
of EGF1 and EGF2 (a,b). The four metal ions
(orange spheres) at the bottom of the
propeller, the metal ion at the genu (orange)
and the ADMIDAS ion (magenta) are also
shown in both (a) and (b). The dotted line in
calf 2 (in [a] and [b]) represents the
disordered loop containing the proteolytic
cleavage site. A short dotted line also
connects PSI and the hybrid domains (visible
in [b]). N and C indicate the amino and
carboxyl terminus, respectively. βTD, β-tail
domain. Adapted and reproduced with
permission from Xiong et al., Science 2001,
294:339-345 [22]. Copyright [2002]
American Association for the Advancement
of Science.
(a)
(b)
Propeller
βA
N
genu
Thigh
Hybrid
PS1
N
EGF1
EGF2
Calf-1
EGF3
EGF4
Calf-2
βTD
C C
C
C
100 Å
Integrin shape and domain composition
The purified extracellular segment of the αA-lacking
integrin αVβ3 was used to derive its crystal structure in the
presence of Ca2+ [22••] and Mn2+ [23••]. In solution, this
ectodomain binds to its physiological ligands — vitronectin,
fibronectin (FN) and fibrinogen (FB) — in a manner comparable to that of the active membrane-bound receptor [83].
In the crystal structure, the αV and β3 subunits join
together to form a head sitting on two legs that are both
flexed at the ‘knees’ (Figure 1a). The integrin is stabilised
in this ‘bent’ conformation by crystal contacts from symmetryrelated molecules which, given their nature, are not expected
to occur as such in vivo (reviewed in [45•]). When the structure
is straightened at the knees, it resembles the familiar
shape of integrins revealed in EM images (Figure 1b).
The integrin α subunit comprises the predicted amino-terminal
seven-bladed β-propeller, which forms part of the head
region. This is followed by three domains: a C2-set
immunoglobulin-like ‘thigh’ domain and two similar β sandwich domains, calf-1 and calf-2, all three forming the α ‘leg’.
The α knee (genu) lies at the junction between the thigh and
calf-1 domains. The proteolytic cleavage site found in most
α subunits is located within a loop in the calf-2 domain, not
visible in the structure and therefore presumably disordered.
The β subunit contains eight domains. A βA domain
contacts the top of the α subunit’s propeller, thus forming
100 Å
the αβ heterodimer. βA and the propeller resemble,
respectively, the Gα and Gβ subunits of G proteins, and
contact each other in a strikingly similar manner [84]. βA
projects from an I-set immunoglobulin-like ‘hybrid’
domain, so named because it is assembled from amino
acids that lie on either side of βA in the primary sequence.
Both the βA and hybrid domains contribute to the head
region. The β leg section is formed of six domains: an
amino-terminal PSI (plexins, semaphorins and integrins
[85]) domain lies at the base of the hybrid domain and is
probably linked to the first of four epidermal growth factor
(EGF) domains by a disulphide bond. The electron density
of PSI, EGF1 and EGF2 is weak, suggesting that these
domains are disordered. The β genu lies in this region.
EGF3 and EGF4 each contains a core of six cysteine
residues linked in a Cys1–Cys3, Cys2–Cys4, Cys5–Cys6
pattern, and a major and minor β strand, all typical of
tandem EGF domains [86]. The minor strand and an epitope
for a monoclonal antibody are lacking in a bacterially
expressed EGF3 from the β2 subunit [87•,88], indicating
that proper folding of the EGF domains requires expressing
them in tandem. An interdomain disulphide bond links
EGF3 and EGF4; by analogy, we suggest that disulphide
bonds link EGF1 to EGF2 and EGF2 to EGF3 ([40•];
JP Xiong et al., unpublished data). Linkage of consecutive
integrin EGF domains via interdomain disulphide bonds
may be necessary to help stabilise the minor CD β sheet
and the interface between tandem EGFs, as revealed in
the EGF3/4 interface of the crystal structure. It is interesting
644
Cell-to-cell contact and extracellular matrix
Figure 2
(a)
(b)
(c)
N215
D217 S121
P219
S144
S123
D126
Ligand- and metal-ion-binding sites in αA and
βA domains. (a) Surface representation of the
RGD ligand-binding site in the head section
of αVβ3. The ligand arginine-binding pocket
(red) is located in the αV propeller (dark grey)
and the aspartic acid binding pocket (blue) in
βA (light grey). The ligand peptide is shown as
a ball-and-stick model (in this and subsequent
figures carbons are shown in green, amides in
blue and oxygens in red). The two visible
Mn2+ ions at MIDAS and ADMIDAS (in cyan
and magenta, respectively, in all figures) are
shown. (b,c) Metal ions at the ligand-binding
interface in βA (b) and αA ([c], from CD11b).
Coordinating residues (single letter code) are
shown as ball-and-stick. The ligand acidic
residue is gold. In addition to the ligand
aspartate, the metal ion (cyan) in the βA
MIDAS is coordinated directly with the
hydroxyl oxygens of Ser121 and Ser123 and
with one carboxylate oxygen from Glu220; the
carboxyl oxygens of Asp119 and Asp251 are
within 6 Å of the metal ion and probably
mediate additional contacts through water
molecules, as in liganded αA (where
equivalent residues are labelled) (c). The
metal ions at ADMIDAS (magenta) and at
LIMBS (grey in all figures) are only found in
liganded βA; their coordinating residues are
labelled. Water molecules are labelled ‘ω’;
hydrogen bonds and metal ion coordination
are represented with dashed red lines.
S142
D158
E220
ω
T209
ω
D127
D119
D140
D251
D242
Current Opinion in Cell Biology
to note that a calcium ion bridge replaces this interdomain
cysteine bridge in the calcium-binding EGF (cbEGF)
variety, for essentially the same purpose [86]. The existence
of a disulphide bridge between EGF1 and -2 and EGF2
and -3 remains controversial however [87], until a highresolution structure of this region in the intact heterodimer
is determined. The β leg ends with a novel β-tail domain
(βTD), which has a limited resemblance to the papain
inhibitor cystatin C [89].
motif. One cation is found in βA at a site adjacent to
MIDAS (ADMIDAS); it is coordinated by the oxygenated
sidechains of Asp126–Asp127 from the α1 helix, and the
carbonyl oxygens of Ser123 (from the A-α1 loop [connecting
the A strand and α1 helix]) and Met335 (from the top of
α7, the F-α7 loop) Metal ions are not found at MIDAS,
however. A sixth cation site is located at the α genu. There
is no metal ion at the centre of the propeller.
The unliganded structure contains six cation-binding sites
present in three regions that are occupied by either Ca2+ or
Mn2+, depending on which metal ion is present in the
crystallisation solution (Figure 1). As predicted, four cation
sites are found at the bottom of the propeller, but each is
ligated within a β-hairpin loop rather than an EF-hand
The structure of αVβ3 in complex with a cyclic RGD was
determined following soaking of the peptide ligand EMD
121974 into pre-formed αVβ3 crystals generated in the
presence of either Ca2+ or Mn2+ [23••]. A strong electron
density for the peptide was found in the presence of Mn2+,
allowing a structure determination of the Mn2+-bound
Structure of αVβ3 integrin in complex with RGD
Coming to grips with integrin binding to ligands Arnaout, Goodman and Xiong
integrin–ligand complex. RGD fits into a crevice between
the propeller and βA domains (Figure 2a) of the bent αVβ3
conformation, and makes extensive contacts with both.
The ligand arginine sidechain inserts into a groove formed
loops D3A3 (the peptide segment connecting strands D3
and A3, between blades 2 and 3) and D4A4 (between
blades 3 and 4) of the propeller [23••], and is held in place
by a bidentate salt bridge to Asp218 at the bottom of the
groove (from the D4A4 loop) and another salt bridge to
Asp150 (from the D3A3 loop) at the rear. The hydrophobic
portion of the arginine sidechain is sandwiched between
the Tyr178 (from the B3C3 loop) and Ala215 (from the
D4A4 loop) sidechains that form the walls of the groove
[23••]. The uppermost portion of the ligand arginine is
exposed to solvent. The glycine residue makes hydrophobic
interactions with the αV subunit, the most critical of which
is with the carbonyl oxygen of Arg216 [23••].
One carboxylate group from the aspartic acid ligand
contacts βA in a strikingly similar manner to the liganded
glutamic acid in the open form of αA (Figure 2b,c). The
ligand aspartic acid contacts a metal ion at an equivalent
MIDAS motif, and is stabilised by additional contacts
with the βA residues Tyr122, Arg214 and Asn215. The βA
MIDAS is formed of the oxygen-containing sidechains of
Asp–X–Ser–X–Ser (Asp119 in the β3 subunit), an invariant
glutamic acid in βA (Glu220 in the β3 subunit, corresponding
to the invariant threonine in αA domains), and by an aspartic
acid residue (Asp251 in the β3 subunit). Interestingly,
Asp251 is present in the same loop that continues to form
the major interface with the propeller. Thus, ligand binding
may effect protein movements at the αβ interface.
645
state, the MIDAS face tightens as the A-α1 loop draws
closer to α2–α3 loop, allowing the MIDAS ion to coordinate
residues from both. These movements appear to be initiated
by the top of the α1 helix moving closer to MIDAS, with
the ADMIDAS moving in concert. The ligand-specificity
region of β3 also approaches the ligand, probably as a result
of a new salt bridge between Asp179 and Arg214 in βA.
Several epitopes for activating and inhibitory monoclonal
antibodies have been mapped to these regions of βA
(reviewed in [40•]), underscoring the functional relevance
of the observed structural changes.
The above movements in A-α1 and α2–α3 loop found in
the peptide-bound βA are very similar in magnitude and
direction to those seen when αA is also bound to a peptide
ligand [46]. In αA, movement of A-α1 loop forces a buried
phenylalanine at the top of the α7 helix into solvency, thus
driving a 10 Å downward movement of this helix. By
contrast, the top of α7 (the F-α7 loop) in βA is tugged
through the ADMIDAS cation to α1. This ionic bond
(lacking αA domains), together with the quaternary
contact of βA with the hybrid domain, are likely to account
for the minimal movement of the α7 helix when βA is
liganded. The dramatic displacement of α7 in the active
state of the αA domain may be a special feature introduced
to regulate ligand-binding activity in the native αAcontaining integrin (see below).
Small quaternary changes are observed when αVβ3 is
occupied by RGD: The propeller and βA domains move
closer together at the peptide-binding site. In addition, the
propeller undergoes a small rotation at the propeller/thigh
interface [23••]. It is possible that additional and/or larger
changes take place when integrins bind to larger or multivalent ligands in their microenvironments.
Comparisons of the MIDAS face in unliganded and liganded
αVβ3 clarifies why the MIDAS ion is absent in the
unliganded state: Glu220 infringes on MIDAS (forming
hydrogen bonds with Asp119) preventing a metal ion from
binding in the unliganded state. In the presence of ligand,
Glu220 has moved out sufficiently to allow access of the
metal ion to MIDAS. The metal ion at ADMIDAS remains
in the liganded structure; its coordination changes somewhat, however, in a way that links it more intimately to
MIDAS (the MIDAS residue Asp251 replaces the Met335
in providing the sixth coordination site at ADMIDAS, as the
α1 helix moves closer to MIDAS in the complex). Finally, a
third metal ion site in βA is created in the liganded state:
only 6 Å away from MIDAS, this ligand-associated metalbinding site (LIMBS) is formed by the other carboxylate
oxygen of Glu220, the sidechains of Asp158, Asn215 and
Asp217 and the carbonyl oxygens of Asp217 and Pro219, all
conserved in βA domains. LIMBS holds Glu220 at a
comfortable distance from the MIDAS pocket, thus permitting
stable binding of the MIDAS cation. LIMBS also adds
structural stability to the ligand-binding surface.
The tertiary and quaternary changes observed in the
αVβ3–RGD complex are clearly related to the presence of
ligand; Mn2+ alone induces none of these changes, although
it may facilitate them. One interpretation of these findings is
that these structural changes are ligand-induced and therefore
represent some of those associated with outside–in signalling.
An alternative interpretation is that the integrin ectodomain
exists in low- and high-affinity states (even in the αVβ3
crystals, where the mobile portions are free from symmetryrelated crystal contacts), one of which (the high-affinity form)
is selected/stabilised by RGD [23••]. Inside–out signals may
shift the equilibrium in favour of this state. In this scenario,
inside-out (activation) is primarily triggered through protein
movements in the β subunit. This is consistent with the facts
that three of the β domains that are linked to activation are
disordered in the structure and are presumably flexible, and
that the vast majority of activating monoclonal antibodies
map to the β subunit (reviewed in [3]).
Tertiary and quaternary changes are observed in the
liganded structure. Tertiary changes is largely confined to
βA and quaternary to the αβ interface. In the liganded
Definition of the ligand-binding site in the αA-lacking
integrin αVβ3 has helped in developing a tantalising
αA as an endogenous integrin ligand
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Cell-to-cell contact and extracellular matrix
Figure 3
(a)
(b)
Current Opinion in Cell Biology
hypothesis [90••] of the relationship between the αA
domain and the core integrin structure. It is well established that αA is necessary and sufficient for ligand
binding [47,63]. In the holoreceptor, however, αA is
dependent on an intact βA MIDAS in ligand binding
[61,62]. αA emanates from the D3A3 loop of the propeller,
which forms part of the ligand-binding pocket in liganded
αVβ3 [23••], indicating a close proximity of the bottom
part of αA to the βA MIDAS. Moreover, the three-residue
and 14–17-residue linkers on the amino and carboxyl
termini of αA, respectively, are solvent-exposed, as judged
by their accessibility to monoclonal antibodies [63,91].
The carboxy-terminal linker contains an invariant glutamic
acid near its amino terminus that follows the α7 helix of
αA. As a result of the 10 Å downward movement of the α7
helix in active αA, this invariant glutamic acid can directly
coordinate the MIDAS cation, thus acting as a formal
ligand (Figure 3a,b). It is contained within a consensus
Ser/Ala–Leu/Ile–Glu–Gly (S/AL/IEG) sequence. In the
whole receptor, we suggest that this glutamic-acid–cation
bridge forms the core of an interface between αA and βA
that locks αA in the open ligand-competent state. When
this link is severed (by artificially introduced mutations),
αA-integrins lose their ability to bind physiological
(activation-dependent) ligands [90••,92], but maintain
their ability to recognise activation-independent ligands
[90••]. Activation by inside–out signals (as judged by binding
of activation-sensitive monoclonal antibodies that map to the
β leg region) is unaffected when the glutamic-acid–cation
bond is broken [90••], suggesting that activation of αA
occurs secondary to inside–out activation of βA.
Lengthening the reach and enriching the ligand profile by
adding an extra domain in some integrins are thus achieved
by utilising the activation and signalling properties inherent
to the basic integrin structure.
A structure model showing a portion of the
head region of the αA-containing integrin
CD11b/CD18 in (a) the low- and (b) the
high-affinity state. αA is connected to the
propeller by two flexible linkers (yellow). In the
low-affinity state, αA is in the closed
conformation; only ADMIDAS but not MIDAS
of βA is occupied by a metal ion. The invariant
glutamic acid sidechain in the carboxyterminal linker at the base of the α7 helix also
points away from βA. In the high-affinity state,
αA is in the open conformation. The 10 Å
downward displacement of α7 allows the
invariant glutamic acid to form a direct bond
with the βA MIDAS ion, stabilising this
conformation. Three of the four metal ions
(orange spheres) at the bottom of the
propeller are also shown. The structures in (a)
and (b) are based on those of unliganded and
liganded αVβ3, respectively, and the closed
(a) and open (b) forms of αA from CD11b.
Insights into ligand binding specificity derived
from structure models of integrins in complex
with physiological ligands
Elucidation of the structure of the αVβ3–RGD complex
has provided insights into the structural basis of ligandbinding specificity. Thus, the absolute requirement for the
RGD (but not KGD, RXD [where X is any amino acid
except glycine] or RGE) motif in all αVβ3 ligands [83,93]
can now be understood in atomic detail. The shorter lysine
cannot form a salt bridge with the propeller’s Asp218 —
any sidechain at the glycine position would introduce
severe clashes with the carbonyl oxygen of βA’s Arg216,
and a glutamic acid instead of aspartic acid in the context
of the RGD peptide cannot be accommodated in the αβ
binding pocket.
The structure of the αVβ3–RGD complex also allows
predictions to be made regarding the number, nature and
location of binding sites for physiologic ligands in other
integrins. It has been amply documented that in addition
to the RGD motif, α5β1 binds to a ‘synergy’ region in
FN, accounting in part for ligand-binding specificity.
Exchanging Gln145–Asp150 from the D3A3 loop joining
blades 2 and 3 of αV propeller with the corresponding
Asp154–Ala159 from α5 is known to endow αV with the
ligand-binding specificity of α5 [70]. The above biochemical
data, together with the precise knowledge of the RGDbinding site and the fact that αV and α5 are highly similar
in sequence (~50% amino acid identity) allows one to build
a structure model of an α5β1–FN complex (Figure 4a). In
this model, the RGD loop in repeat 10 of FN (FN10,
utilising the crystal structure of repeats 7–10 of the human
fragment [16]) can be readily superimposed on the remarkably
similar RGD loop structure of the cyclic peptide bound to
the bent αVβ3 with no clashes (Asp227 of α5, equivalent
to Asp218 of αV, is predicted to form the salt bridge with
Coming to grips with integrin binding to ligands Arnaout, Goodman and Xiong
647
Figure 4
(a)
(b)
FN9
(c)
VCAM-1
D2
FN9
FN10
Arg1379
FN10
FB-γ
VCAM-1
D1
Current Opinion in Cell Biology
Structure models of integrins in complex with physiological ligands.
(a) Human α5β1 in complex with repeats 7–10 of FN (FN7–10); only FN9
and 10 (green) are shown. The synergy residue Arg1379 in FN9 is also
shown. The RGD loop of FN10 is shown in blue. Inset: the whole bent
integrin in complex with FN9–10 is shown, for orientation purposes. The
secondary ligand-binding site in (a), as well as in (b) and (c), involves the
outer (free) side of the integrin’s propeller. (b) Human αIIbβ3 in complex
with FB-γ chain (green); a portion of FB-β (yellow) is shown. The KQAGD
loop is at the carboxyl terminus of FB-γ. (c) Human α4β1 in complex with
the first two domains of VCAM-1 (green). The integrin contacts each ligand
at roughly the same regions (note the similar orientation of the propeller
and βA domains with respect to the ligand in each case). Only a portion of
the integrin head region is shown in (a)—(c). The ions at MIDAS (cyan) and
LIMBS (grey) are seen in these views. Some of the propeller’s metal ions
(orange) are also visible. The models were generated using Modeler 4.0
[113], using the criteria listed in the text.
the ligand Arg). The synergy domain located in repeat 9
of FN (FN9) lies sideways in relation to the α5 propeller,
facing the outer side of blade 3, with the Asp154 sidechain
(from the D3A3 loop of α5) being ~6 Å away from the key
synergy-domain residue Arg1379 of FN9 [94], even
without making further adjustments at the flexible FN9/10
interface [95]. In αIIbβ3, Glu157 from the αIIb propeller
occupies the equivalent position to Asp154 of α5 and may
contribute to the activation-dependent [96] binding of FN
to this receptor. The above model suggests that electrostatic
interactions are involved in binding of the synergy domain
in FN9 to α5β1 and αIIbβ3, in agreement with the
available biochemical data [97]. Interestingly, the small
rotation of the propeller seen in liganded αVβ3, moves the
propeller’s Asp154 of α5 slightly away from the aspartic
acid ligand, helping to optimise the distance between the
RGD and the synergy binding sites in the integrin. It is
also interesting to note that the predicted synergy-binding
region of the propeller is fully accessible in the bent
integrin conformation.
residues 294–314 (the second metal-binding hairpin loop
in blade 5, at the bottom of the αIIb propeller) [98]. The
FB-binding site has been mapped, however, to loops at the top
of the αIIb propeller (Gly183–Gly193 [B3C3 loop of blade
3] [100], Pro145 [D3A3 loop] [101] and Asp224 [D4A4 loop]
[102]) and to the β3 segments containing MIDAS/ADMIDAS/LIMBS residues (Asp119, Ser211–Gly222). Binding of
αIIbβ3 to the ligand-mimetic monoclonal antibodies PAC-1,
LJ-CP3 and OP-G2 requires αIIb residues Val156–Trp162
(from the D3A3 loop) and Glu229–Tyr330 (from the D4A4
loop), in addition to the βA residues Asp179–183 (from the
ligand-specificity BC loop) [103].
Human FB, a 340 kDa dimer of an αβγ trimer, has two RGD
sequences in each α and β chain. Binding of fibrinogen to
αIIβ3 is not mediated by its RGD sequences, however,
but by a Lys–Gln–Ala–Gly–Asp (K406QAGD) sequence at
the carboxyl terminus of its γ chain (FB-γ). Synthetic
peptides corresponding to this region inhibit FB, as well
as FN and von Willebrand factor binding to platelets [98].
FB–αIIβ3 interaction is activation-dependent and is
blocked by RGD or KGD (from the integrin antagonist
barbourin [99]). Biochemical studies found that FB-γ
residues 400–411 (containing KQAGD) crosslinked to
Although it cannot be formally excluded that a binding site
for FB may exist at the propeller’s bottom, the balance of
the biochemical data favours that this site is located at the
αβ interface. It has been suggested that RGD and
KQAGD bind a single site [73], two distinct sites 6.1 nm
apart [74,75,98,104] or two allosterically linked sites [79].
The availability of the crystal structure of the carboxyl
terminus of human FB [105] and a low-resolution model of
the whole FB molecule [106] allows us to build a hypothetical structure model of αIIβ3 in complex with a whole
FB molecule. The KQAGD loop of FB-γ orients with the
lysine and aspartic acid sidechains in opposite directions,
in positions that are largely superimposible, respectively,
on those of arginine and aspartic acid residues in the cyclic
RGD–αVβ3 complex. The ligand Lys406 and Asp410 thus
fit, respectively, into the arginine pocket of αIIb and the
MIDAS pocket in β3 (Figure 4b). Amino-terminally, the
FB-γ globular domain faces the same side of the αIIb
propeller n coordinating the synergy sequence of FN
648
Cell-to-cell contact and extracellular matrix
(outer side of blade 3). Thus aligned, the rest of the native
FB structure displays no clashes or additional contacts with
the integrin. Moreover, it can easily accommodate binding
of a second integrin in the FB dimer, providing the first
atomic model of FB-mediated crosslinking of two integrin
receptors and a structural basis for platelet aggregation.
The present model suggests that FB binds to the same
region as RGD in αIIβ3, although coordination of the
ligand lysine may be somewhat different from that of the
ligand arginine in RGD.
In contrast to other integrins, α4β1 recognises aspartic acid
within a different sequence motif. It binds to VCAM-1
(CD106) through a Gln–Ile–Asp–Ser–Pro (QIDSP) sequence
and to the alternatively spliced type III connecting segment of
fibronectin through a Glu–Ile–Leu–Asp–Val–Pro–Ser–Thr
(EILDVPST) sequence at the carboxyl terminus of CS1
[107,108]. Chemical crosslinking of a Leu–Asp–Val-based
small-molecule inhibitor (in which leucine was replaced with
lysine for crosslinking purposes) identified a single cationdependent binding site in the β1 subunit containing the
DXSXS sequence of MIDAS (Asp130–Leu–Ser–Tyr–Ser of
β1) [68]. CS1 or VCAM-1 effectively competed for binding to
this site. When crosslinking was done using an amino-groupspecific crosslinker with a longer flexible spacer arm (11.4 Å),
the α4 subunit was also labelled [68]. Mutation of Asp130 abolished CS1 and VCAM-1 binding to α4β1 [109]. Swapping the
α4 propeller residues 112–131 (the B2C2 loop in blade 2) or
237–247 (the B4A4 loop and flanking strands of blade 4) with
those in α5 abolished adhesion to VCAM-1 and CS1 [110].
On the basis of these data and the availability of the
crystal structure of the ligand-binding domains 1 and 2 of
VCAM1, a structural model of α4β1 in complex with
VCAM-1 can be built with no substantial clashes
(Figure 4c). In this model, the aspartic acid ligand from
domain 1 (in the QIDSP motif) binds to the metal ion at
MIDAS, while glutamine is accommodated in the arginine
pocket of the α4 subunit glutamine is favoured by the
nature of this pocket in α4. In this model, the α4 propeller
residues 112–131 face one side of domain 2 of VCAM-1,
suggesting their direct involvement in ligand binding. On
the other hand, the α4 residues 237–247 are not predicted
to bind the ligand directly in this model.
Conclusions
The crystal structure of extracellular αVβ3 alone and in
complex with the prototypical ligand RGD has provided
insights into the nature of the tertiary and quaternary
changes that accompany binding of this ligand. New models
based on the αVβ3–RGD structure propose a structural
basis for cation-mediated binding of extracellular physiological ligands such as FN, FB and VCAM-1 to α5β1,
αIIbβ3 and α4β1, respectively. On the basis of these structure models, we propose that ligand binding is located at
two sites in αA-lacking integrins: the first is the RGDbinding pocket at the αβ interface, which can be occupied
by RGD (in many ligands), KQAGD (in αIIbβ3), QIDS
(in α4β1) or S/AL/IEG (in αA, when the integrin contains
the additional αA domain). It is likely that binding at this site
represents the initial, fast and reversible step often described
in many integrin interactions with ligands. The αA MIDAS
face constitutes an alternative site for binding exogenous
ligands in αA-integrins (since the RGD pocket is occupied by
the endogenous ligand αA and is therefore inaccessible).
A second ‘synergy’ site (for some ligands) is predicted along
the outer side of the propeller’s blade 3. This site faces FN9,
the globular head of FB-γ and domain 2 of VCAM-1 of the
respective integrin in our models. Its occupation may
account for the slower and more stable phase of integrin–ligand
interactions [111,112]. Ligand-binding specificity is contributed by both sites (the βA ligand-specificity loop is
considered here to be part of the RGD site). The tertiary
changes observed in liganded αVβ3 may occur in response
to inside–out signals in the native receptors. If the small
ligand-associated rotation of the propeller seen when αVβ3
is bound by RGD is any indication, occupation of the RGD
site may help expose/reorient the synergy site to create an
optimal and stable complimentary interface between the
integrin and physiological ligands. Understanding in
structural terms how such activation-dependent interactions
are translated into specific intracellular responses presents
the next significant challenge.
Acknowledgements
MAA and JPX are supported by grants from the US National Institutes
of Health.
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