Structural basis for distinctive recognition of fibrinogen γC peptide by the platelet integrin αIIbβ3

Hemostasis and thrombosis (blood clotting) involve fibrinogen binding to integrin αIIbβ3 on platelets, resulting in platelet aggregation. αvβ3 binds fibrinogen via an Arg-Asp-Gly (RGD) motif in fibrinogen's α subunit. αIIbβ3 also binds to fibrinogen; however, it does so via an unstructured RGD-lacking C-terminal region of the γ subunit (γC peptide). These distinct modes of fibrinogen binding enable αIIbβ3 and αvβ3 to function cooperatively in hemostasis. In this study, crystal structures reveal the integrin αIIbβ3–γC peptide interface, and, for comparison, integrin αIIbβ3 bound to a lamprey γC primordial RGD motif. Compared with RGD, the GAKQAGDV motif in γC adopts a different backbone configuration and binds over a more extended region. The integrin metal ion–dependent adhesion site (MIDAS) Mg2+ ion binds the γC Asp side chain. The adjacent to MIDAS (ADMIDAS) Ca2+ ion binds the γC C terminus, revealing a contribution for ADMIDAS in ligand binding. Structural data from this natively disordered γC peptide enhances our understanding of the involvement of γC peptide and integrin αIIbβ3 in hemostasis and thrombosis.


Introduction
Integrins are formed from ␣ and ␤ subunits, each with a large extracellular domain and a single, more C-terminal transmembrane domain. The subunits come together in a large interface between the ␣ subunit ␤ propeller domain and the ␤ subunit ␤ I domain to form the ligand-binding head. Other domains form upper and lower legs in each subunit to connect the head to the membrane. Integrins have an overall bent conformation in the low affi nity state, with the bend between the upper and lower legs. Upon activation, integrins extend, and a major reorientation at the interface between the ␤ I and hybrid domains occurs that is linked to remodeling of the ligand-binding site in the ␤ I domain ( Luo et al., 2007 ). The ␤ I domain has three metal ionbinding sites, with a Mg 2+ ion in the central metal ion -dependent adhesion site (MIDAS) fl anked by two Ca 2+ ions, one of which is in a site termed adjacent to MIDAS (ADMIDAS). Structures of ␣ V ␤ 3 and ␣ IIb ␤ 3 bound to cyclic RGD-like peptides show binding across the ␣ subunit ␤ propeller interface with the ␤ subunit I domain. The Asp side chain coordinates the Mg 2+ ion at the ␤ subunit I MIDAS, whereas the Arg side chain binds to Asp residues in the ␣ subunit ␤ propeller domain ( Xiong et al., 2002;Xiao et al., 2004 ).
Early in hemostasis and thrombosis, the integrin ␣ IIb ␤ 3 on platelets is activated and binds to its ligand fi brinogen. Fibrinogen is a dumbbell-shaped molecule ( Fig. 1 a ). Integrin ␣ IIb ␤ 3 binds specifi cally to the distal ends of the dimeric fi brinogen molecule in a natively unstructured region at the C terminus of the ␥ subunit ( ␥ C peptide; Yang et al., 2001 ). The large separation between the two ␣ IIb ␤ 3 -binding sites on fi brinogen of ‫ف‬ 440 Å is well suited for cross-linking of platelets, which results in platelet aggregation and formation of platelet plugs in hemostasis and thrombosis. In a later event in hemostasis, cleavage of peptides from the N termini of the fi brinogen ␣ and ␤ subunits stimulates assembly of fi brinogen into fi brin. Yet later, adjacent fi brinogen molecules within fi brin are cross-linked through their ␥ C peptides by the factor XIIIa transglutaminase ( Hawiger, 1995;Bennett, 2001 ).
One of the best established paradigms in integrin biology is the recognition of protein ligands through Arg-Gly-Asp (RGD) sequences, the majority of which are present within fl exible loop regions. Eight vertebrate integrins, including ␣ IIb ␤ 3 and ␣ V ␤ 3 , recognize RGD sequences in ligands, and crystal structures are beginning to reveal how RGD is recognized, at least in cyclic peptides ( Xiong et al., 2002;Xiao et al., 2004 ). H emostasis and thrombosis (blood clotting) involve fi brinogen binding to integrin ␣ IIb ␤ 3 on platelets, resulting in platelet aggregation. ␣ V ␤ 3 binds fibrinogen via an Arg-Asp-Gly (RGD) motif in fi brinogen ' s ␣ subunit. ␣ IIb ␤ 3 also binds to fi brinogen; however, it does so via an unstructured RGD-lacking C-terminal region of the ␥ subunit ( ␥ C peptide). These distinct modes of fi brinogen binding enable ␣ IIb ␤ 3 and ␣ V ␤ 3 to function cooperatively in hemostasis. In this study, crystal structures reveal the integrin ␣ IIb ␤ 3 -␥ C peptide interface, and, for comparison, integrin ␣ IIb ␤ 3 bound to a lamprey ␥ C primordial RGD motif. Compared with RGD, the GAKQA-GDV motif in ␥ C adopts a different backbone confi guration and binds over a more extended region. The integrin metal ion -dependent adhesion site (MIDAS) Mg 2+ ion binds the ␥ C Asp side chain. The adjacent to MIDAS (ADMIDAS) Ca 2+ ion binds the ␥ C C terminus, revealing a contribution for ADMIDAS in ligand binding. Structural data from this natively disordered ␥ C peptide enhances our understanding of the involvement of ␥ C peptide and integrin ␣ IIb ␤ 3 in hemostasis and thrombosis.
Whether RGD and ␥ C peptides bind to distinct or similar sites on ␣ IIb ␤ 3 is controversial. Several studies show competition for the same binding site ( Lam et al., 1987 ;Santoro and Lawing, 1987;Bennett, 2001 ). Interestingly, although ␥ C and RGD peptides were found to cross-compete, a ␥ C dodecapeptide was photo -cross-linked only to the ␣ IIb subunit, whereas a RGD hexapeptide was cross-linked to both the ␣ IIb and ␤ 3 subunits ( Santoro and Lawing, 1987 ). Binding has been described of RGD and ␥ C peptides to distinct, nonoverlapping, allosterically linked sites ( Hu et al., 1999 ). RGD and RGD-like peptides have also been described to bind to distinct sites on ␣ IIb ␤ 3 ( Cierniewski et al., 1999 ). Furthermore, ␥ C peptide was reported to cross-link to an ␣ IIb site ( D ' Souza et al., 1990 ) that is distal from the binding site for RGD shown in crystal structures ( Xiao et al., 2004 ).
Because of the biological and clinical importance of the ␥ C peptide in hemostasis and thrombosis, there has been great interest in determining its biologically relevant integrin-bound However, the ␥ C region of fi brinogen lacks an RGD motif. Studies on synthetic peptides show specifi c binding of ␣ IIb ␤ 3 to the ␥ C C-terminal dodecapeptide, a gradual loss of activity when the dodecapeptide is truncated from its N terminus, and complete loss of activity when the C-terminal pentapeptide is removed. The minimal peptide with inhibitory activity was found to be the C-terminal pentapeptide, QAGDV ( Kloczewiak et al., 1984( Kloczewiak et al., , 1989. Furthermore, removal of the ␥ C QAGDV sequence but not of two RGD sequences in fi brinogen abolishes binding to ␣ IIb ␤ 3 ( Farrell et al., 1992 ;Holmback et al., 1996 ). Thus, although RGD peptides, including those corresponding to sequences in fi brinogen, can inhibit binding of ␣ IIb ␤ 3 to fi brinogen, the ␥ C peptide is the biologically important recognition site for ␣ IIb ␤ 3 within fi brinogen. In contrast to ␣ IIb ␤ 3 , integrin ␣ V ␤ 3 on endothelial cells binds to a distinct site containing an RGD motif in the fi brinogen ␣ subunit ( Cheresh et al., 1989 ;Smith et al., 1990;Doolittle and Kollman, 2006 ). This cooperative interaction between ␣ IIb ␤ 3 and ␣ V ␤ 3 integrins allows the platelet/fi brinogen thrombus formed by activation of ␣ IIb ␤ 3 to  ( Yang et al., 2001 ) with ␣ , ␤ , and ␥ subunits in green, red, and blue, respectively, and natively unstructured C-terminal regions of the ␣ and ␥ subunits ( Yang et al., 2001;Doolittle and Kollman, 2006 ) that contain RGD and KQAGDV sequences shown schematically as dashed lines. (b) The C-terminal sequence of the ␥ subunit in different species. Basic and acidic residues that are important for binding to fi brinogen as shown here for human and implicated for other species are shown in red. Gln and Lys residues known (human and lamprey; Strong et al., 1985 ) or implicated (other species) to mediate fi brinogen cross-linking catalyzed by factor XIIIa transglutaminase are underlined. Sequence accession (GI) numbers of the relevant fi brinogen splice variants are 70906437, 73977998, 19527078, 61098186, 45384500, 120145, and 120143. brinogen ␥ subunit sequences in a diverse range of vertebrates (fi brinogen is found only in vertebrates) revealed that RGD is found in ␥ C in frog and lamprey ( Fig. 1 b ). This observation, which, to our knowledge, is previously unremarked and therefore was a surprise to us, suggests an obvious evolutionary pathway from promiscuous integrin recognition of RGD in the primordial jawless vertebrate the lamprey to monospecifi c integrin recognition of non-RGD sequences in most higher vertebrates. Therefore, for comparison to human ␥ C, we also soaked crystals with chimeric deca-or dodecapeptides containing the RGD sequence present in lamprey ␥ C ( Fig. 1 b ). Structures were determined at 2.4 -2.8-Å resolution ( Table I ), and previous structures with antagonists were rerefi ned to lower R free ( Table II ). The structures contain the ␤ propeller domain in the ␣ IIb subunit; the I, hybrid, plexin-semaphorin integrin, and integrin EGF-like (I-EGF) domain 1 in the ␤ 3 subunit; 15 carbohydrate residues; and bound ligand and Fab. The structures all have an open headpiece (i.e., with the hybrid domain swung out) and the ␤ I domain in the high affi nity state ( Xiao et al., 2004 ) The ␥ C peptide binds at the interface between the ␣ IIb ␤ propeller and ␤ 3 I domains ( Fig. 2 a ). The binding site overlaps with, but is more extensive on both the ␣ and ␤ subunits, than previously described for cyclic RGD peptides ( Xiong et al., 2002;Xiao et al., 2004 ). The contacts on the ␤ 3 subunit include not only MIDAS but also a novel water-mediated coordination of the ␥ C C terminus with ADMIDAS, as described in more detail in the next section. The ␥ C peptide contacts with ␣ IIb lie in a groove between two long loops that connect blades ( ␤ sheets of the ␣ IIb ␤ propeller) 2 and 3 and blades 3 and 4. The cap subdomain of the ␤ propeller ( Xiao et al., 2004 ) forms one side of this groove ( Fig. 2 b ). In the region of the ␥ C peptide N terminal to residue 404, extension along the groove conformation. However, in multiple crystal structures of fi brinogen and its C-terminal ␥ subunit fragment, the ␥ C peptide is disordered Spraggon et al., 1997 ;Yee et al., 1997;Yang et al., 2001 ;Kostelansky et al., 2002 ). Crystals of the ␥ C peptide fused to lysozyme or glutathione S -transferase reveal different conformations ( Donahue et al., 1994 ;Ware et al., 1999 ) that are unrelated to the integrin-bound conformation described here. Nuclear magnetic resonance measurements of ␥ C interaction with ␣ IIb ␤ 3 provided limited information ( Mayo and Fan, 1996 ). In the absence of appreciation of the features important for specifi c recognition of ␥ C by ␣ IIb ␤ 3 , pharmaceutical development of antithrombotics began with compounds with RGD or KGD motifs. Subsequent synthesis and screening then focused on obtaining compounds that were selective for ␣ IIb ␤ 3 compared with other integrins that recognize RGD, such as ␣ V ␤ 3 and ␣ 5 ␤ 1 ( Scarborough and Gretler, 2000 ). Two RGD-based drugs, a small molecule, tirofi ban, and a cyclic peptide, eptifi batide, are currently used clinically to prevent thrombosis, and their complex structures with ␣ IIb ␤ 3 have been determined ( Xiao et al., 2004 ).
In this study, we investigate how the ␥ C peptide and a related peptide containing the RGD sequence present in lamprey ␥ C bind to ␣ IIb ␤ 3 . The results reveal the biologically important interface between ␥ C peptide and ␣ IIb ␤ 3 and unexpected features of the integrin recognition of ligands, including a direct role for ADMIDAS in ligand recognition.

Overall complex structure
We soaked human ␥ C deca-and dodecapeptides into ␣ IIb ␤ 3 headpiece/Fab crystals ( Xiao et al., 2004 ). Examination of fi - and F calc ( h ) are the observed and calculated structure factors, respectively. No I/ cutoff was applied. R free is the R value obtained for a test set of refl ections consisting of a randomly selected 5% subset of the data set excluded from refi nement. a Determined with RAMPAGE ( Lovell et al., 2003 ). b These numbers correspond to the last resolution shell.
complexes with the chimera peptides show how an RGD peptide unconstrained by cyclization binds to an integrin and provide the best comparison with the ␥ C peptide. The RGD tripeptide adopts a highly extended conformation across the integrin ␣ / ␤ intersubunit interface ( Fig. 3 b ). The Arg and Asp side chains extend in opposite directions, and the backbone in between is also extended. The conformation of the ligand backbone is stabilized as it crosses the interface between the ␣ IIb and ␤ 3 subunits by two previously unremarked water molecules with strong density that are held in place by hydrogen bonds to the side chain of ␣ IIb Asp-232 and backbone of ␤ 3 Ala-218 and that hydrogen bond to the carbonyl oxygen of the Arg of RGD ( Fig. 3 b ). The Arg side chain of RGD forms a charged hydrogen bond to ␣ IIb residue Asp-224 and also forms a hydrogen bond to an ␣ IIb backbone carbonyl. The Asp side chain of RGD directly coordinates to the MIDAS Mg 2+ cation and is further satisfied by three hydrogen bonds to the ␤ 3 backbone and one to the side chain of ␤ 3 Asn-215. The same geometry at the Asp is seen for all ␣ IIb ␤ 3 complexes, which are in the high affi nity state ( Fig. 3, a -c ). Only one of the four Asp hydrogen bonds are seen at the Asp of the ␣ V ␤ 3 complex ( Fig. 3 d ) because it has a closed headpiece, and the two backbone nitrogens of the ␤ 1 -␣ 1 loop have not moved within hydrogen bonding distance; furthermore, the side chain of Asn-215 should probably be fl ipped. By reducing the loss of peptide entropy upon binding, cyclization can increase affi nity and is therefore widely adopted in drug development. Eptifi batide is a seven-residue L peptide that is cyclized with a disulfi de bond, contains homoarginine-Gly-Asp in place of RGD, and is in clinical use for prevention is blocked by contact of ␥ C residue Leu-402 with a neighboring molecule in the crystal lattice, and residues 400 -403 have weak electron density and extend toward solvent. Therefore, we limit our structural analysis to ␥ C octapeptide residues 404 -411, which show strong electron density in omit maps, intimately contact ␣ IIb ␤ 3 ( Fig. 2 and Fig. 3 a ), and have essentially identical conformations in crystals soaked with ␥ C pep tides 400 -411 and 402 -411 ( Table I ).
Surface representations of the binding site show not only the groove in which the ␥ C peptide lies but two deeper pockets within this groove ( Fig. 2 b ). The penultimate residue in the ␥ C peptide, Asp-410, buries its side chain in a pocket at the MIDAS Mg 2+ ion in the ␤ 3 I domain. The surface at the MIDAS Mg 2+ ion is not visible in this view, which is in contrast to the surface at the ADMIDAS Ca 2+ ion ( Fig. 2 b , purple), which is much more solvent exposed. The other deep pocket, on the ␣ IIb side of the interface, is occupied by Lys-406 of the ␥ C peptide ( Fig. 2 b ). This is the same pocket that is occupied by the Arg side chain of RGD peptides. Fig. 3 compares the binding modality of the ␥ C peptide ( Fig. 3 a ) to that of RGD-like peptides ( Fig. 3, b -d ). We fi rst will describe the RGD peptide-binding modality and then return to ␥ C and compare it with RGD. The A408R substitution yielding the lamprey-like RGD motif in the chimera peptide ␥ C ( Fig. 1 b ) results in retention of ␣ IIb ␤ 3 antagonist activity ( Kloczewiak et al., 1989 ). In the chimera ␥ C peptide, the Arg-408 side chain extends directly into the ␣ IIb -binding pocket ( Fig. 3 b ) and displaces Lys-406, which binds in this pocket in the ␥ C peptide ( Fig. 3 a ).
In contrast to previous integrin structures with cyclized peptides ( Xiong et al., 2002;Xiao et al., 2004 ), the ␣ IIb ␤ 3 and F calc ( h ) are the observed and calculated structure factors, respectively. No I/ cutoff was applied. R free is the R value obtained for a test set of refl ections consisting of a randomly selected 5% subset of the data set excluded from refi nement. a Determined with RAMPAGE ( Lovell et al., 2003 ).
Asp moiety of ␥ C has a conformation identical to that of Arg-Gly-Asp, and the carbonyl oxygen of Ala-408 hydrogen bonds to the two water molecules held in place by ␣ IIb Asp-232 at the interface with the ␤ 3 subunit in a geometry identical to that seen for the Arg and homoarginine carbonyl oxygens of RGD and eptifi batide ( Fig. 3, a -c ). Indeed, the C ␤ atoms of Ala-408 of ␥ C and the Arg of RGD occupy identical positions ( Fig. 3, a and b ). The critical difference between the ␥ C peptide -and RGD peptide -binding modalities is the backbone turn at ␥ C residue Gln-407, which orients the N-terminal portion of the ␥ C peptide into the groove formed by the long loops that connect ␣ IIb ␤ propeller blades 2 and 3 and blades 3 and 4 ( Fig. 3 a ). The backbone turn at ␥ C Gln-407 enables the ␥ C Lys-406 side chain to enter the ␣ IIb binding pocket from a markedly different location than the Arg side chain of RGD ( Fig. 3, a and b ). The last few atoms of the Lys-406 side chain turn to approach ␣ IIb Asp-224 from a similar direction as Arg ( Fig. 3, a and b ). A 15-fold loss of potency upon substitution of Lys-406 with Arg ( Kloczewiak et al., 1989 ) is explicable by the inability of the planar guanido group of Arg to similarly turn. Hydrophobic ␣ IIb residues and Phe-231 line the pocket and contact the aliphatic portion of the ␥ C Lys-406 side chain and the Ala-408 side chain.
of thrombosis ( Fig. 3 c ). Eptifi batide binds similarly to RGD, and the carbonyl oxygen of its homoarginine residue similarly hydrogen bonds to the waters held in place by ␣ IIb Asp-232 ( Fig. 3 c ). However, cyclization pins back the homoarginine C ␣ and N atoms so that the C ␥ atom of homoarginine is in a similar position to the C ␤ atom of Arg, explaining the requirement for the onecarbon longer homoarginine side chain ( Fig. 3, b and c ). Cilengetide, an ␣ V ␤ 3 antagonist, is a fi ve-residue peptide that contains RGD, N -methyl-Val, and d -Phe ( Fig. 3 d ). The Arg of cilengitide forms a charged hydrogen bond to ␣ V residue Asp-218. Cilengetide is cyclized through its peptide backbone in a considerably smaller ring than epitifi batide. Tight cyclization, which is important to prevent binding to ␣ IIb ␤ 3 ( Scarborough and Gretler, 2000;Gottschalk and Kessler, 2002 ), pulls the Arg backbone back away from the ␣ V subunit and helps place the Arg side chain further above the subunit than the Arg or homoarginine of ␣ IIb ligands, as appropriate for the shallower ␣ V binding pocket ( Fig. 3 d ).
The fi brinogen ␥ C peptide complex with ␣ IIb ␤ 3 reveals that ␥ C residue Lys-406 forms a charged hydrogen bond to ␣ IIb residue Asp-224 ( Fig. 3 a ). Therefore, ␥ C Lys-406 is functionally equivalent to the Arg of RGD ( Fig. 3 b ) and the homoarginine of eptifi batide ( Fig. 3 c ). Furthermore, the Ala-Gly- ␥ C residues Gly-404, Ala-405, and Lys-406 fi ll the groove between adjacent ␤ propeller blades and form backbone -backbone and backbone -side chain hydrogen bonds to ␣ IIb residues Asp-159 and Ser-226 ( Fig. 3 a ). ␥ C residues Gly-404 and Ala-405 also seal the binding pocket for the Lys-406 side chain, with ␥ C residue Gly-404 forming a backbone hydrogen bond to the ␥ C Lys-406 side chain.

ADMIDAS coordination
An important and unexpected observation is that the free carboxyl group of the ␥ C C-terminal residue Val-411 coordinates the ␤ 3 I domain ADMIDAS calcium ion through an intermediate water molecule with strong density ( Fig. 3 a ). The contribution of this coordination to ligand binding is demonstrated by the fi nding that amidation of the ␣ -carboxyl group decreases by sixfold the potency of ␥ C peptides in inhibiting fi brinogen binding to ␣ IIb ␤ 3 ( Kloczewiak et al., 1989 ). An identical ADMIDAS coordination is seen with the chimera QRGDV peptide ( Fig. 3 b ). The signifi cance of this observation, the notable similarity in backbone positions of Val-411 of the ␥ C and chimera peptides, and the equivalent residue in eptifi batide, Trp-5 ( Fig. 3, a -c ), is discussed in the next section.

␥ C peptide by ␣ IIb ␤ 3
Our structures of ␥ C peptides bound to ␣ IIb ␤ 3 reveal the interface that is biologically important for the binding of fi brinogen to activated integrin ␣ IIb ␤ 3 on platelets. This binding event, in turn, leads to cross-linking of platelets by fi brinogen and the formation of platelet plugs in hemostasis and thrombosis. Whereas Arg-Gly-Asp binds to eight different integrins, the fi brinogen ␥ C peptide binds only to ␣ IIb ␤ 3 .
Our ␥ C peptide complex with ␣ IIb ␤ 3 reveals the basis for binding of platelet ␣ IIb ␤ 3 and endothelial ␣ V ␤ 3 to distinct sites in fi brinogen, enabling these integrins to have complementary rather than competitive functions in hemostasis ( Fig. 1 a ; Cheresh et al., 1989 ;Smith et al., 1990 ). The interface on the integrin ␣ subunit occupied by the ␥ C peptide is much larger than that occupied by RGD. ␥ C residues Gly-404, Ala-405, Lys-406, and Ala-408 all interact with ␣ IIb . In contrast, in the linear RGD chimera peptide, only Arg-408 binds to the ␣ IIb subunit. This much more extensive interface with ␥ C enables specifi c differences between ␣ IIb and other integrin ␣ subunits to be recognized as good contacts made by ␣ IIb and clashes made by ␣ V . The shallower and narrower binding site in the ␣ V subunit precludes binding of the KQAGDV moiety using the ␣ IIb ␤ 3 -bound conformation. For example, ␣ V Ala-215 and Asp-218 would both clash with KQAGDV. These results show why the ␥ C peptide does not bind to ␣ V .
In contrast, RGD does bind to ␣ IIb ␤ 3 , and, therefore, the selectivity of ␣ IIb ␤ 3 for the ␥ C site over RGD in fibrinogen ( Cheresh et al., 1989 ;Smith et al., 1990 ) must refl ect a higher affi nity for ␥ C. Fibrinogen lacking the ␥ C QAGDV pentapeptide fails to bind ␣ IIb ␤ 3 and results in bleeding disorders in mice ( Farrell et al., 1992 ;Holmback et al., 1996 ). This emphasizes Figure 3. Binding of the linear ␥ C and RGD chimera peptides and comparison with cyclic peptides. Ligand, ␣ subunit, and ␤ subunit are shown in green, light blue, and beige, respectively, with O, N, and S atoms in red, blue, and yellow, respectively. Water molecules are red spheres. Ca 2+ ions are gold spheres, and Mg 2+ ions (a -c) or Mn 2+ ions (d) are silver spheres. Fc-Fo omit maps after refi nement in the absence of ligands and the displayed water molecules are contoured at 3 . The complexes are ␣ IIb ␤ 3 (a -c) with ␥ C dodecamer (a) or chimera decamer (b) peptides, disulfi decyclized eptifi batide peptide (c), or ␣ V ␤ 3 with backbone-cyclized cilengitide peptide (d; Xiong et al., 2002 ). One-letter code is used for residues except for homoarginine (Har). Residue C7 in eptifi batide (c) is amidated. ␣ IIb -Asp-224 than to ␣ V -Asp-218. The fi nal drug developed from barbourin, eptifabitide, has a homoarginine side chain in place of lysine ( Fig. 3 c ). This drug binds very similarly to RGD ( Fig. 3 b ) and quite differently from the ␥ C peptide, in which the Lys side chain enters the ␣ IIb binding pocket from a completely different direction ( Fig. 3 a ). Thus, drug design starting from KGD ironically resulted in an RGD-like antagonist with a binding modality very different from KQAGDV.
The ␥ C complex structure provides new insights for the development of second generation integrin antagonists. Mimicking the novel route into the ␣ IIb binding pocket adopted by the Lys of the KQAGDV motif should enable development of new classes of highly specifi c ␣ IIb ␤ 3 antithrombotics. Furthermore, mimicking the novel interaction with the ADMIDAS metal ion is attractive for improving the specifi city and affi nity of antagonists to a wide range of integrins.
The fi nding that the ␥ C peptide -binding site completely overlaps the RGD peptide -binding site defi nitively resolves the long-standing controversy about whether these peptides compete for binding to the same site or bind to distinct sites ( Lam et al., 1987 ;Santoro and Lawing, 1987;D ' Souza et al., 1990 ;Hu et al., 1999 ;Bennett, 2001 ). Furthermore, our studies explain previous observations of photo -cross-linking of the ␥ C dodecapeptide peptide to only the ␣ IIb subunit and of an RGD hexapeptide to both the ␣ IIb and ␤ 3 subunits, with cross-competition by RGD and ␥ C peptides, respectively ( Santoro and Lawing, 1987 ). Both peptides appeared to have the radiolabeled, photoactivatable reagent attached to their N-terminal ␣ -amino groups. The exclusive labeling of ␣ IIb by the reagent attached to ␥ C His-400 is consistent with the position of Gly-404 in our structures in the ␣ IIb ␤ propeller groove and also with further extension in the N-terminal direction in this groove. In contrast, labeling of both the ␣ IIb and ␤ 3 subunits by the reagent attached to the residue before the Arg of RGD is consistent with the position of the corresponding Gln-407 residue at the interface between the ␣ IIb and ␤ 3 subunits in the chimera deca-and dodecamer structures.

A role for ADMIDAS in ligand binding
Previous crystal structures and mutation of MIDAS-coordinating residues have shown that the MIDAS metal ion has a direct role in ligand binding ( Luo et al., 2007 ). In contrast, the ADMIDAS metal ion has been shown to have a regulatory role in ligand binding. During conversion from the low affi nity, closed conformation to the high affi nity, open conformation of the integrin headpiece, remodeling of the ␤ 1 -␣ 1 loop that coordinates to the ADMIDAS metal ion shifts the position of this metal ion by 3 Å ( Xiao et al., 2004 ). Ca 2+ and Mn 2+ compete for binding to ADMIDAS, resulting in inhibition or stimulation of ligand binding, respectively ( Chen et al., 2003 ). We note that physiologically and in the current structures, Ca 2+ is present at ADMIDAS ( Xiao et al., 2004 ) and that coordination is pentagonal bipyramidal, with seven oxygen ligands as typically seen for Ca 2+ . ADMIDAS mutations augment ligand binding by integrins ␣ 4 ␤ 7 and ␣ L ␤ 2 ( Chen et al., 2003( Chen et al., , 2006 and, in contrast, inhibit ligand binding by ␣ IIb ␤ 3 and ␣ 5 ␤ 1 ( Bajt and Loftus, 1994 ;Mould et al., 2003 ). In this study, we fi nd that in addition to its regulatory role, ADMIDAS can also directly contribute to ligand the absolutely critical role of the ␥ C peptide in recognition by ␣ IIb ␤ 3 ; however, it does not mean that the ␥ C peptide is responsible for all of ␣ IIb ␤ 3 ' s affi nity for fi brinogen. Indeed, ␣ IIb ␤ 3 has 100-fold higher affi nity for fi brinogen than for the ␥ C octapeptide ( Kloczewiak et al., 1983( Kloczewiak et al., , 1984. Mutagenesis studies show that the binding site on the ␣ IIb ␤ propeller domain includes, but is larger than, the ␥ C octapeptide -binding site defi ned here ( Kamata et al., 2001 ;Xiao et al., 2004 ). The 10E5 Fab binds within this region of the ␣ IIb ␤ propeller that is important in fibrinogen binding and blocks binding of fi brinogen but not the octapeptide to ␣ IIb ␤ 3 . Because the N terminus of the octapeptide extends toward the 10E5 Fab -binding site ( Fig. 2 ), it is tempting to speculate that regions N terminal to the ␥ C peptide, including well-folded ␥ C module residues 144 -392, also bind to ␣ IIb ␤ 3 and, together with the ␥ C peptide, account for the higher biological affi nity of ␣ IIb ␤ 3 for fi brinogen than for RGD peptides. Such other regions of the ␥ C domain may also account for the retention of clot-retraction activity by fi brinogen deleted in the QAGDV pentapeptide ( Holmback et al., 1996 ). Integrin binding or inhibition of clot retraction has been demonstrated using portions of ␥ C N terminal to KQAGDV made in Escherichia coli ( Medved et al., 1997 ;Yokoyama et al., 1999;Podolnikova et al., 2003 ); however; E. coli ␥ C fragments also express integrin-binding sites that are not present on native fi brinogen ( Akakura et al., 2006 ).
Multiple crystal structures of fi brinogen and its fragments show that ␥ chain residues 404 -411 are disordered and that residues 393 -403, when ordered, completely differ in conformation from one crystal lattice to another Spraggon et al., 1997 ;Yee et al., 1997;Yang et al., 2001 ;Kostelansky et al., 2002 ). Previous intense and creative efforts using carrier protein-driven crystallization ( Donahue et al., 1994 ;Ware et al., 1999 ) failed to reveal the biologically relevant conformation of the ␥ C peptide.
Our structures reveal the conformation that ␥ C residues 404 -411 adopt when bound to ␣ IIb ␤ 3 . Although not inconsistent with the previous conclusion that QAGDV represents a minimal recognition unit in ␥ C, our structure resolves the conundrum that QAGDV lacks a basic residue by demonstrating that Lys-406 fulfi lls a function similar to the Arg in RGD and that the KQAGD motif in ␥ C occupies the same site as RGD. Residues Gly-404 and Ala-405 also appear to have an important role in sealing the binding pocket for Lys-406 in the ␣ IIb groove.
In the absence of information on how the ␥ C peptide binds ␣ IIb ␤ 3 , development of the two currently approved small molecule ␣ IIb ␤ 3 therapeutics, tirofi ban and eptifi batide, proceeded from RGD and barbourin, respectively ( Scarborough and Gretler, 2000 ). Barbourin is a disintegrin with two highly unusual properties for a disintegrin: specifi city for ␣ IIb ␤ 3 and KGD in place of the RGD motif ( Scarborough et al., 1991 ). Lysine has a side chain with C ␤ , C ␥ , C , and N atoms. The Arg side chain has C ␤ , C ␥ , and C ␦ atoms and a guanido group with N , N 1 , N 2 , and C atoms. The three N atoms of Arg give it versatility for hydrogen bonding. Although the Arg side chain readily forms charged hydrogen bonds through different guanido nitrogens to ␣ IIb -Asp-224 in Fig. 3 b and ␣ V -Asp-218 in Fig. 3 d , it is easy to imagine how a Lys in a similar position might better form a hydrogen bond to in RGD is conserved as a hydrophobic residue and suggest that the three-residue insertion in bony fi shes can be accommodated as a longer turn between the Lys and this hydrophobic residue.
The presence of an RGD motif in both lamprey and frog and the presence of a KQ(A/V)GDX motif in warm-blooded vertebrates and a KQFGG(I/L)GDI motif in bony fi shes ( Fig. 1 b ) raises the possibility that evolution from the recognition of Arg to Lys occurred more than once. A plausible stepping stone in this evolutionary process is provided by the function of Lys in fi brinogen cross-linking. In lamprey as well as in higher vertebrates, fi brinogen is cross-linked within the ␥ C peptide by the factor XIIIa transglutaminase ( Strong et al., 1985 ). Lys-406 is crosslinked to Gln-398 or Gln-399 in human, and the Lys N-terminal to the RGD in lamprey is cross-linked to one of the more N-terminal Gln residues ( Strong et al., 1985 ). Xenopus laevis appears similar to lamprey, with Lys and Gln residues N-terminal to the RGD. In contrast, humans, other warm-blooded vertebrates, and bony fi shes resemble one another by all having the lysine that is known (human) or implicated (other species) in binding to ␣ IIb ␤ 3 as the same lysine that is known (human) or implicated (other species) in cross-linking. In ␥ C peptides with RGD motifs, the Lys that functions in cross-linking could later have evolved a second function in specifi c recognition of a platelet integrin, providing an evolutionary stepping stone from RGD-based recognition of fi brinogen by multiple integrins to Lys-based recognition of fi brinogen by a specifi c integrin on platelets or thrombocytes. Whether there is any more signifi cance to the dual function of Lys-406 in human fi brinogen ␥ is unclear. However, there would be little, if any, competition between these two functions of Lys-406 because fi brinogen binding to platelets occurs much earlier in the clotting cascade than fi brin formation, and the fi brinogen that contributes to fi brin formation is in great excess over that bound to platelet integrin ␣ IIb ␤ 3 .

Materials and methods
The ␥ C dodecapeptide was obtained from the American Peptide Co., and other peptides were synthesized and verifi ed by mass spectrometry by the biopolymer facility at the Department of Biological Chemistry and Molecular Pharmacology at Harvard Medical School (Boston, MA). Crystals of the ␣ IIb ␤ 3 headpiece bound to 10E5 Fab in 11% polyethylene glycol 3350, 0.7 M magnesium acetate, and 0.1 M cacodylate, pH 6.5 ( Xiao et al., 2004 ), were cryoprotected with a solution containing 15% polyethylene glycol 3350, 0.7 M magnesium acetate, and 0.1 M imidazole, pH 6.5, plus 5% step increases of glycerol concentration to a fi nal 20% glycerol, and then 50 μ M of synthetic peptides was added in the fi nal cocktail. Crystals were soaked for 4 d at 4 ° C before freezing in liquid nitrogen. Diffraction data were collected with beamline 19ID (Advanced Photon Source) and processed with program suite HKL2000 ( Otwinowski and Minor, 1997 ). As there is little difference in the dimensions of the unit cell compared with nonpeptide-soaked crystals, the previously determined structure of the ␣ IIb ␤ 3 headpiece complexed with 10E5 Fab (1TXV; 2.7 Å ) was directly subjected to rigid body refi nement program CNS ( Brunger et al., 1998 ) using the diffraction data. Electron density calculated using phases from the refi ned structure clearly showed the presence of the peptides. The peptide structures were built with program O and subjected to iterative cycles of model rebuilding in O and refi nement using CNS (version 1.1) as previously described ( Xiao et al., 2004 ).
Subsequent rebuilding with Coot and refi nement with REFMAC5 ( Murshudov et al., 1997 ) resulted in structures with unusually low R free ( Tables I and II ), which may be attributed to multiple factors. Regions for rebuilding were identifi ed using multiple verifi cation tools in Coot to detect structural defects supplemented with improved defi nition of Ramachandran outliers, clashes, and improbable rotamers using MolProbity ( Davis et al., 2007 ). Rebuilding fi rst focused on the higher resolution 2.4-and 2.6-Å ␥ C binding. The ␥ C C-terminal COOH group coordinates the ADMIDAS Ca 2+ ion through water with strong electron density. The importance of this interaction is demonstrated by the sixfold loss in inhibitory potency of ␥ C peptides when the C terminus is amidated.
It should be noted that the amidation experiment only demonstrates that coordination to a charged carboxyl oxygen is stronger than to a carbonyl oxygen. Both types of oxygens form direct and indirect water-mediated coordinations to Ca 2+ ( Harding, 2001 ). Therefore, the importance of ADMIDAS coordination may extend beyond fi brinogen to include ligands with RGD sequences that are not followed by the C-terminal residue. In eptifi batide, the Arg-Gly-Asp-Trp-Pro-Cys sequence places a Pro in the same position as in the Arg-Gly-Asp-Ser-Pro sequence of fi bronectin; this Pro, the bulky Trp, and cyclization place constraints on backbone conformation. Nonetheless, the Trp carbonyl oxygen in eptifi batide is in a position very similar to that of one of the two ␣ -carboxyl oxygens of the ␥ C and chimera peptide Val-411 residues ( Fig. 3, a -c ). In the absence of the constraints in the cyclic peptide, this carbonyl could move slightly and form a water-mediated coordination to the ADMIDAS similarly to the ␥ C and RGD chimera peptide Val-411 carboxyl group. Water with weak density may be in a position to make this coordination in the eptifi batide complex structure ( Fig. 3 c ). Furthermore, the carbonyl oxygen of the amidated Cys two residues after the Trp forms a water-mediated coordination to the ADMIDAS ( Fig. 3 c ). Water-mediated ADMIDAS coordination to RGD ligands provides a plausible explanation for the fi nding that mutation of ADMIDAS-coordinating residues inhibits RGD-dependent binding of ␣ 5 ␤ 1 to fi bronectin ( Mould et al., 2003 ) and binding of ␣ IIb ␤ 3 to the peptide GRGDSP ( Bajt and Loftus, 1994 ), although ADMIDAS mutations also appear to favor the inactive conformation of ␣ 5 ␤ 1 ( Mould et al., 2003 ). We propose that the stimulatory effect of ADMIDAS mutations on ligand binding by integrins that do not recognize RGD, ␣ 4 ␤ 7 ( Chen et al., 2003 ), and ␣ L ␤ 2 ( Chen et al., 2006 ) refl ects the regulatory role of ADMIDAS and that the inhibitory effect of ADMIDAS mutations on ligand binding by integrins that recognize RGD (or KQAGDV), ␣ IIb ␤ 3 ( Bajt and Loftus, 1994 ), and ␣ 5 ␤ 1 ( Mould et al., 2003 ) refl ects a direct role of ADMIDAS in ligand recognition, in addition to a regulatory role. This proposal requires testing with further integrinligand crystal structures.

Evolution of the KQAGDV motif from an RGD motif in fi brinogen
Fibrinogen fi rst evolved in vertebrates ( Jiang and Doolittle, 2003 ). The signifi cance of ADMIDAS coordination by the C-terminal carboxyl group of the fi brinogen ␥ subunit is emphasized by the fact that in vertebrates, the position of the C-terminal carboxyl group relative to the penultimate Asp residue has been invariant for the last 450 million years ( Fig. 1 b ; Strong et al., 1985 ). Thus, the interaction with ADMIDAS of the C-terminal carboxyl group fi rst evolved in jawless vertebrates in the context of an RGD motif and was maintained when warm-blooded vertebrates evolved the KQ(A/V)GD motif and bony fi shes evolved the KQFGG(I/L)GD motif in place of the RGD motif ( Fig. 1 b ). Our structures also explain why in fi brinogen the residue that takes the place of the Arg chimera deca-and dodecapeptide structures. These structures were refi ned in parallel so that rebuilding could focus on problems identifi ed by verifi cation tools or discrepancies between the model and electron density that were shared between two independent datasets. One of the shared discrepancies was a clash at ␣ IIb residue 282 accompanied by a lack of density for the putative Ala-282 C ␤ atom; checking of the nucleotide sequence used for ␣ IIb expression and RefSeq entry gi88758615 reveals Gly at this position compared with Ala in UniProt entry P08514. Rebuilding of the Fab was aided by comparison with multiple high resolution V H , C HI , V L , and C L domains that were found by BlastP search of the protein database and were individually superimposed on the 10E5 Fab. In the four ␥ C and chimera ␥ C peptide complexes, Fab residue Asn-157 in the light chain appears to have largely undergone deamidation with conversion to isoaspartic acid. This residue is in an Asn-Gly sequence, the sequence known to be most susceptible to deamidation ( Aswad et al., 2000 ), and previous protein chemistry on the mouse light chain peptide containing this sequence revealed heterogeneity, suggesting chemical rearrangement ( Svasti and Milstein, 1972 ). The previously determined headpiece structures ( Table II ) did not show evidence for isoaspartic acid, which is consistent with deamidation during prolonged storage of the Fab before preparation of the more recent crystals ( Table I ).
Although isoaspartic acid has been built in one case ( Esposito et al., 2000 ), we did not do so because of the diffi culty of implementing the correct restraints for an isopeptide bond in the peptide backbone and the mixture of products, including L-and D-aspartic acid, that result from deamidation ( Aswad et al., 2000 ). The Xpleo server was used to guide rebuilding of several loops that were diffi cult to build manually ( van den Bedem et al., 2005 ). Although not built in our previous headpiece structures ( Xiao et al., 2004 ), I-EGF domain 1 residues 436 -461 are clearly present, although they are less well ordered than other domains. The last disulfi de-bonded loop of I-EGF1, residues 462 -472, either was disordered or was removed by carboxypeptidase A, which stopped at Arg-461. Arg is known to be resistant to digestion by carboxypeptidase A ( Ambler, 1967 ). Compared with our previous headpiece structures ( Xiao et al., 2004 ), carbohydrate linkages were corrected to those known to occur in high mannose N-linked glycans; three cis-prolines were added, and one was removed. TLS refi nement in REFMAC5 used groups determined with the TLS Motion Determination server ( Painter and Merritt, 2006 ) and contributed a 2 -3% drop in R free .
The ␥ C dodecamer and decamer structures and the fi ve previously deposited ␣ IIb ␤ 3 headpiece 10E5 Fab complex structures ( Xiao et al., 2004 ) were then rerefi ned starting with the ligands in the previous models and the headpiece-Fab moiety from the refi ned chimera ␥ C decamer complex 2.4-Å structure described in Table I . Ligand topology and parameter cif fi les were created by the PRODRG2 server ( Schuttelkopf and van Aalten, 2004 ). The cacodylate and eptifi batide ligands are unchanged. Use of correct chirality and fl ip of the peptide bond relative to the thienothiophene moiety in the L-739758 ligand and rotations around the sulfonamide N atoms in L-739758 and tirofi ban result in better fi ts to density and hydrogen bonding to ␣ IIb ␤ 3 . The R free of the fi ve previously deposited structures is decreased by 2.1 -5.1%. ␣ IIb residue 123 appears to be a true Ramachandran outlier; other outliers are in regions of poor density. All coordinates and structure factors have been deposited with the PDB. The unusually low R free of the deposited structures is consistent with quality assessment by the MolProbity server ( Davis et al., 2007 ), which returns MolProbity scores in the 100th percentile for all nine structures (where 100th percentile is the best) compared with other deposited structures at similar resolutions.