Integrin α5β1 is a key receptor for the extracellular matrix protein fibronectin. Antagonists of human integrin α5β1 have therapeutic potential as anti-angiogenic agents in cancer and diseases of the eye. However, the structure of the integrin is unsolved and the atomic basis of fibronectin and antagonist binding by integrin α5β1 is poorly understood. In the present study, we demonstrate that zebrafish α5β1 integrins do not interact with human fibronectin or the human α5β1 antagonists JSM6427 and cyclic peptide CRRETAWAC. Zebrafish α5β1 integrins do bind zebrafish fibronectin-1, and mutagenesis of residues on the upper surface and side of the zebrafish α5 subunit β-propeller domain shows that these residues are important for the recognition of the Arg-Gly-Asp (RGD) motif and the synergy sequence [Pro-His-Ser-Arg-Asn (PHSRN)] in fibronectin. Using a gain-of-function analysis involving swapping regions of the zebrafish integrin α5 subunit with the corresponding regions of human α5 we show that blades 1–4 of the β-propeller are required for human fibronectin recognition, suggesting that fibronectin binding involves a broad interface on the side and upper face of the β-propeller domain. We find that the loop connecting blades 2 and 3 of the β-propeller, the D3–A3 loop, contains residues critical for antagonist recognition, with a minor role played by residues in neighbouring loops. A new homology model of human integrin α5β1 supports an important function for D3–A3 loop residues Trp157 and Ala158 in the binding of antagonists. These results will aid the development of reagents that block integrin α5β1 functions in vivo.
- RGD motif
- synergy region
The interactions of integrin receptors with extracellular matrix macromolecules are critical for development, responses to injury and normal tissue homoeostasis [1–3]. Integrin α5β1 is a fibronectin receptor found on a wide variety of cell types. Two distinct sites in fibronectin are involved in binding to integrin α5β1; the first lies in the tenth type III repeat (3Fn10) and encompasses the Arg-Gly-Asp (RGD) motif , whereas a second, weaker, interaction site is found in the ninth type III repeat (3Fn9) and includes the sequence Pro-His-Ser-Arg-Asn (PHSRN; the so-called ‘synergy’ sequence) [5–7]. These two sequences are separated by 30–40 Å (1 Å=0.1 nm) in the tertiary structure of this region of fibronectin .
Integrin α5β1 is also highly expressed in pathological situations, for example on many tumour cell types and on activated endothelial cells during the formation of tumour vasculature [9–13]. Moreover, there is evidence that integrin α5β1–fibronectin interactions play a key role in diseases of the eye that involve neovascularization [14–18]. Hence, there is a great deal of interest in the development of antagonists of integrin α5β1 for therapeutic use [19,20].
Presently, no high resolution structural information has been obtained for integrin α5β1–ligand interactions. However, crystal structures of the closely related integrins αVβ3 and αIIbβ3 [21–23] show that the RGD motif binds in a pocket formed by loops on top of the α subunit β-propeller domain and the A-domain of the β subunit (known as βA or βI). Small molecule antagonists bind in the same pocket , thereby preventing ligand recognition. Homology models of integrin α5β1 have been constructed using the structure of αVβ3 bound to the antagonist cilengitide [24,25] as a template; these models have aided the rational design of high-affinity α5β1 antagonists such as JSM6427 . Phage display technology has also been used to identify high-affinity peptide ligands for integrin α5β1 such as the disulfide-bridged cyclic peptides Cys-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys (CRRETAWAC), Cys-Arg-Gly-Asp-Gly-Phe-Cys (CRGDGFC) and Cys-Arg-Gly-Asp-Gly-Trp-Cys (CRGDGWC) [26,27]. These peptides have been shown to interact with loops on the upper face of the α5 subunit β-propeller domain [28–30]. However, limited data are currently available concerning the precise mode of antagonist binding by integrin α5β1. In addition, there is controversy concerning the mechanism of fibronectin recognition, specifically whether the synergy sequence binds directly to the α5 subunit  or indirectly supports the binding of RGD to the integrin . Hence, there is a pressing need to further define the mechanisms of ligand binding by integrin α5β1.
Previously, we identified two close homologues of the mammalian integrin β1 subunit in zebrafish, β1-1 and β1-2 (also known as β1a and β1b) . In the present study, we show that the zfα5 (zebrafish α5 integrin subunit) can form functional heterodimers with both zfβ1 (zebrafish β1 integrin subunit) or with the huβ1 (human β1 integrin subunit); however, all of these zfα5 integrins show no binding to human fibronectin or human α5β1 antagonists. This lack of ligand binding enabled us to use a gain-of-function approach to identify the regions of the α5 subunit required for interactions with human fibronectin and antagonists. We demonstrate that a loop region between the second and third blades of the β-propeller (D3–A3 loop) plays a key role in antagonist binding, but a much more extensive region of the β-propeller is necessary for recognition of the whole fibronectin ligand. A new homology model of integrin α5β1, based on the αIIbβ3–tirofiban structure , supports a prominent function for residues at the apex of the D3–A3 loop in the binding of antagonists.
Additional experimental details can be found in the Supplementary Experimental section available at http://www.BiochemJ.org/bj/424/bj4240179add.htm.
Peptides GACRRETAWACGA (CRRETAWAC), GACRRETADACGA (CCRETADAC) and GCRGDSPCG (cyclic-RGD) were purchased from Peptide 2.0. Peptides were cyclized by oxidation as described previously . Small molecules JSM6427 and JSM6406 were provided by Jerini AG.
Cloning and mutagenesis
A full-length zfα5 cDNA clone was a gift from S. Koshida (Okazaki, Japan). Full-length cDNA clones for two zebrafish β1 subunits, β1-1 and β1-2, were obtained as previously described . Recombinant soluble versions of human, zebrafish and chimaeric integrins, fused to the hinge region and CH2 and CH3 domains of the human IgGγ1 chain, were made essentially as before , using the complete extracellular domain of the α5 subunit and the headpiece of the β1 subunit (PSI, hybrid and βI domains). For cloning into the pEE12.2Fc vector , an internal HindIII site in zfα5 was altered from AAGCTT to AAACTT by overlap-extension PCR, creating a silent mutation. Constructs were verified by DNA sequencing. The amino acid sequence of the zfα5 construct was identical with that predicted from the UniProt entry B3DJJ0 (although note that the signal peptide should be 32, not 14, amino acid residues in length) with the exception that Ser488 in the mature sequence was replaced by asparagine. This substitution lies outside the β-propeller domain and so is unlikely to affect function. The sequences of β1-1 and β1-2 were identical with those reported previously (GenBank® accession numbers DQ149101 and DQ149102). Constructs containing the head region of the β1-1 or β1-2 (equivalent to the previously reported β1TR construct ) were made by cloning a PCR product containing residues Gln21–Pro475 of β1-1 (Gln1–Pro455 in the mature sequence) or Gln18–Pro472 of β1-2 (Gln1–Pro455 in the mature sequence) fused with a murine antibody leader sequence [34,39] into the pV.16hFc vector. The corresponding huα5 (human α5 integrin subunit) and huβ1TR–Fc clones  were a gift from J. Askari (University of Manchester). Blade-swapping chimaeras were created by overlap-extension PCR using huα5 and zfα5-Fc constructs as templates. The W1–W3 blade swap contains residues Phe1–Asp228 of huα5, the W1–W4 swap contains residues Phe1–Met281 of huα5 and the W2–W4 swap contains residues Ile70–Met281 of huα5. Loop-swapping mutations in zfα5 were created using overlap-extension PCR. To determine the sequences for swapping, alignment of zebrafish and human sequences was performed using the ClustalW sequence alignment program (http://www.ebi.ac.uk/Tools/clustalw2/index.html). Site-directed mutagenesis was carried out using the QuikChange II XL kit (Stratagene) or by overlap-extension PCR. Oligonucleotides for cloning and mutagenesis were supplied by Eurogentec; oligonucleotides for alanine-scanning mutations were a gift from S. Holley (Yale University, New Haven, CT, USA) or from Eurogentec. Wild-type huα5–Fc and huβ1TR–Fc constructs were prepared as described previously .
A cDNA encoding a cell-binding fragment of FN-1 (zebrafish fibronectin-1), the 3Fn6–10 region including the constitutively included EIIIB domain , was obtained by reverse transcription of mRNA from 3-day-old zebrafish embryos as described previously . This animal experiment was performed in accordance with U.K. Home Office regulations. The sequence of the cDNA was identical to that predicted from the UniProt entry B3DGZ1 for residues Thr1093–Thr1632, but differed from the published sequence  at the 3Fn8/9 boundary. The cDNA was cloned into the pCEP-PU expression vector, which encoded a C-terminal FLAG-tag. This construct was used for transfection of 293-EBNA cells (see below).
Transient transfection of CHO (Chinese-hamster ovary) cells
CHO-L761h cells  were maintained in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% (v/v) fetal bovine serum, 2 mM glutamine and 1% non-essential amino acids (Invitrogen). Plates (six-well) of sub-confluent CHO-L761h cells were transfected with 1 μg of β1 construct and 1 μg of α5 construct/well using LipofectaminePLUS™ reagent (Invitrogen) according to the manufacturer's instructions. After 6 days, culture supernatants were harvested by centrifugation at 1000 g for 5 min.
All the reagents for SDS/PAGE were from Invitrogen except for the protein markers that were obtained from Bio-Rad Laboratories. Aliquots of cell culture supernatants were run on 3–8% NuPAGE™ Tris/acetate gels (Invitrogen) under non-reducing conditions, transferred on to nitrocellulose membranes and blotted with anti-(human Fc) horseradish peroxidase-conjugated antibody (Stratech Scientific). Bands were visualized using UptiLight™ enhanced chemiluminescence reagent (Cheshire Biosciences).
Solid-phase ligand-binding assays
The 50 kDa cell-binding domain of human fibronectin (3Fn6–10; ‘50K’) was produced in Escherichia coli as described previously . The 70 kDa cell-binding fragment of FN-1 (‘70K’) was produced by transient transfection of 293-EBNA cells using Lipofectamine™ PLUS reagent according to the manufacturer's instructions. Cells were cultured in DMEM or Ham's F12 with GlutaMAX™ (Invitrogen) containing 0.1 unit/ml penicillin and 10 μg/ml streptomycin for 7 days. The 70K fragment was purified from the cell culture medium using anti-FLAG M2-affinity gel chromatography (Sigma–Aldrich).
For receptor–ligand-binding assays, the binding of biotinylated 50K or 70K fibronectin fragments to recombinant receptors (captured from cell culture supernatants using goat polyclonal anti-human Fc antibody) was measured in the presence of 1 mM Mn2+ at room temperature (20 °C) as previously described . Measurements obtained were the means±S.D. for four replicate wells. Background binding to wells coated with BSA alone was subtracted from all measurements.
In all assays comparing the binding of fibronectin fragments with wild-type and mutant receptors, the level of ligand binding to mutant receptors was normalized relative to that of wild-type zfα5β1-1 by measuring the binding of the anti-(human Fc) horseradish peroxidase-conjugated antibody to a parallel set of replicate wells for each receptor . Each experiment shown is representative of at least three separate experiments. Binding or inhibition curves were fitted using global optimization by simulated annealing (GOSA-fit, http://www.bio-log.biz).
Apparent Ki values for inhibitors were calculated using the formula: where Kd is the apparent affinity of ligand binding to recombinant integrin α5β1. In inhibition assays the concentration of ligand was 2–3 nM and inhibitor concentrations were 0.1–100 μg/ml for CRRETAWAC and 0.1–300 nM for JSM6427 (prepared by serial dilution).
Alignment of α5 with αV and αIIb, and β1 with β2 and β3, was performed using ClustalW2. This alignment was identical with that of Xiong et al.  within the β-propeller and βI/A domains. Homology modelling of the α5 β-propeller domain, and the β1 head region, was carried out using Modeller 9  using the PDB 2VDR structure of αIIbβ3  as a template, except for residues Arg220–Tyr233 of α5 where the PDB 1L5G structure of αV  was used as a template. Lowest-energy models were chosen, and selected loops were refined using Modeller 9. Final quality of the model was assessed using PROCHECK ; 87.8% of side chains were in the most favoured regions, 10.9% in additional allowed regions, 0.5% in generously allowed and 0.8% in disallowed regions of the Ramachandran plot. Residues in disallowed or generously allowed regions of the plot did not contribute to the ligand-binding pocket. DS Vizualizer 2.0 (Accelerys Software) was used to visualize the model. Docking of a fragment of JSM6427 into the ligand-binding pocket was performed with the constraints of placing the 2-amino group in a suitable position for hydrogen bonding to the carboxy group of Asp227 on the α5 subunit and orientation of the carboxy group of JSM6427 towards the MIDAS (metal-ion-dependent adhesion site) cation on the β subunit. The program PyMOL (http://www.pymol.org) was used to display the model.
Characterization of zebrafish α5 integrins
Constructs containing the complete extracellular domain of the zfα5 subunit and the head region of the β1-1 and β1-2 subunits fused to the hinge region and CH2 and CH3 domains of the Fc region of the human IgGγ1 chain were created. These constructs were chosen because expression of the head and leg regions of the α subunit, together with the head region of the β subunit, has previously been shown to be sufficient to create a constitutively active integrin . The zfα5 construct (zfα5–Fc) was transiently co-expressed with β1-1–Fc, β12–Fc or huβ1TR–Fc  constructs in CHO-L761h cells. Recombinant integrins were partially purified from the cell culture medium and the formation of heterodimers was examined using Western blotting. The results (Figure 1A) showed that in each case a predominant band at approx. 240 kDa was observed, corresponding to the expected mass of an α,β–Fc heterodimer. Under reducing conditions two predominant bands were observed at approx. 50 and approx. 100 kDa, results consistent with the expected sizes of the α5–Fc and the β1–Fc subunits respectively (results not shown). Together, these results show that zfα5 can form heterodimers not only with β1-1 and β1-2, but also with huβ1.
Zebrafish α5 integrins bind to zebrafish but not human fibronectin
A recombinant fragment of FN-1, containing the α5β1-binding sites (type III repeats 6–10) was produced in mammalian cells. As this fragment has an apparent molecular mass of approx. 70 kDa by SDS/PAGE, we refer to this protein as ‘70K’. The 70 K protein has a similar synergy sequence to human fibronectin (PPSRS in place of PHSRN) and an identical RGD motif-containing loop (see Supplementary Figure S1 available at http://www.BiochemJ.org/bj/424/bj4240179add.htm). To test whether the recombinant integrins were functional, binding of biotin-labelled 70K protein to recombinant zfα5 integrins was examined in solid-phase assays. Ligand binding was measured in the presence of 1 mM Mn2+ in order to fully activate the receptors . The results (Figure 2A) showed that each receptor bound strongly to 70K in a concentration-dependent and saturable manner. 70K binding could be inhibited by EDTA or a cyclic RGD peptide, showing that the interaction was specific (see Supplementary Figure S2 available at http://www.BiochemJ.org/bj/424/bj4240179add.htm). Apparent affinities could be calculated from the binding curves (see Supplementary Table S1 http://www.BiochemJ.org/bj/424/bj4240179add.htm) and these results showed that the affinity of zfα5β1-1 for FN-1 was approx. 3-fold higher than that of zfα5β1-2. Surprisingly, zfα5huβ1 bound 70K with the highest affinity. We also examined whether the zebrafish integrins could bind to an equivalent recombinant fragment of human fibronectin (50K ). The results (Figure 2B) showed that there was essentially no binding of zfα5β1-1 or zfα5β1-2, and only very low binding of zfα5huβ1 to 50K.
Human α5 can form functional heterodimers with zebrafish β1 subunits
As described above, we found that zfα5 could associate with huβ1. We next tested whether the huα5 subunit could form heterodimers with the two zebrafish β1 subunits. The huα5–Fc construct was co-transfected with huβ1–Fc, β1-1–Fc or β1-2–Fc constructs in CHO-L761h cells. Cell culture supernatants were analysed for the formation of heterodimers as described above. The results showed a predominant band of approx. 260 kDa in each case, the expected size of the heterodimer (Figure 1B). The apparent molecular mass of the huα5 heterodimers was observed to be slightly greater than that of the corresponding zebrafish receptors (Figure 1B), probably because of the presence of additional glycosylation sites in huα5 . Heterodimer formation was also assessed using antibodies against the β-propeller domain of the α5 subunit (see Supplementary Figure S3 available at http://www.BiochemJ.org/bj/424/bj4240179add.htm). The epitopes of these monoclonal antibodies are only expressed when the β-propeller assumes its native fold, and this folding is dependent on the association of α5 with the βI domain . We found that anti-α5 β-propeller epitopes were expressed to a similar level for huα5β1-1, huα5β1-2 and huα5huβ1 receptors. No binding of these antibodies was observed when cells were transfected with the α5 subunit alone. Collectively, these results demonstrate that the huα5 subunit can form heterodimers with zebrafish β1-1 and β1-2.
We next examined whether the huα5 integrins could bind to the human 50K fibronectin fragment. The results (Figure 2C and see Supplementary Table S1) showed that all three receptors bound with high affinity to 50K. This binding was specific as it could be fully inhibited by EDTA or cyclic RGD peptide (Supplementary Figure S2B). We then tested whether the human receptors could bind to the zebrafish 70K protein (Figure 2D). We found that the huα5 integrins could interact with this zebrafish fibronectin fragment but with markedly lower affinity than with the human protein (see Supplementary Table S1).
In summary, we found that zfα5 integrins interacted strongly with the FN-1 fragment but not with the human protein, whereas the huα5 integrins bound preferentially to the human fibronectin fragment. To further support these findings, we investigated if zfα5β1-1–Fc could bind to purified human plasma fibronectin in an inverted assay in which fibronectin fragments or whole fibronectin are coated on to a 96-well plate and the integrin is in the solution phase. The results (see Supplementary Figure S4A available at http://www.BiochemJ.org/bj/424/bj4240179add.htm) showed that zfα5β1-1–Fc bound well to 70K but not to 50K or huFN. In contrast, huα5β1-1–Fc bound poorly to 70K but strongly to 50K or huFN (Supplementary Figure S4B). We also found that zebrafish fibroblasts, which express zfα5β1-1, spread very well on the 70K zebrafish protein but poorly on the 50K human protein (see Supplementary Figure S5 available at http://www.BiochemJ.org/bj/424/bj4240179add.htm), suggesting that our in vitro result is relevant to the function of cell-surface integrins.
Alanine-scanning mutagenesis identifies residues in loops on the side and upper surface of the zebrafish α5 subunit β-propeller involved in the binding of fibronectin
We next examined whether the mechanism of fibronectin binding to zfα5 integrins was similar to that previously reported for huα5β1. In huα5, residues around Phe187 and Asp227 are predicted to be important for the binding of the RGD motif [21,42], whereas Tyr208 and Ile210 appear to play a role in recognition of the synergy sequence . On the basis of an alignment with huα5 (see Supplementary Figure S6 available at http://www.BiochemJ.org/bj/424/bj4240179add.htm) alanine mutations were made in zfα5 residues in either the RGD motif (F183A, D224A, D225A) or proposed synergy sequence (Y204A, L206A)-binding sites . These residues lie in loops on the upper face or side of the β-propeller domain (the corresponding residues in huα5 are Phe187, Asp227, Asp228, Tyr208 and Ile210). CHO cells were transfected with wild-type α5 or α5 mutants together with β1-1–Fc, and recombinant integrins were tested for binding to 70K. The results (Table 1 and see Supplementary Figure S7 available at http://www.BiochemJ.org/bj/424/bj4240179add.htm) showed that the Tyr204 or Leu206 putative synergy sequence-binding site mutations reduced the affinity of ligand binding approx. 30-fold. A previously described mutation of Tyr204 to asparagine (Y204N) causes a loss-of-function phenotype in vivo  and had a more severe effect on ligand recognition than the Y204A mutation. The F183A and D224A putative RGD motif-binding site mutations essentially abrogated ligand recognition. The D225A mutation had an intermediate effect (Table 1 and see Supplementary Figure S7) and a control mutation K154A in a neighbouring loop had no effect on ligand binding (results not shown). In summary, the same residues appear to be important for recognition of fibronectin by both zfα5 and huα5 integrins.
Zebrafish α5 integrins are not inhibited by human α5 antagonists
We next investigated whether there were any differences between the human and zebrafish integrins in their sensitivity to human α5β1 integrin antagonists. For these assays we used the small molecule JSM6427  (see Supplementary Figure S8 available at http://www.BiochemJ.org/bj/424/bj4240179add.htm) and the cyclic peptide CRRETAWAC . The ability of these compounds to inhibit binding of 50K to huα5 integrins or 70K to zebrafish integrins was tested. The results (Figure 3) showed that all the huα5 receptors were potently inhibited by JSM6427 and CRRETAWAC but these reagents had little or no effect on the zebrafish heterodimers. In control experiments, the compound JSM6406 (an inactive derivative of JSM6427) and the peptide CRRETADAC, in which the essential tryptophan residue is replaced by aspartate, were ineffective for blocking 50K binding to huα5β1-1 (see Supplementary Figure S9 available at http://www.BiochemJ.org/bj/424/bj4240179add.htm). Apparent Ki values calculated from the inhibition curves for JSM6427 and CRRETAWAC (see Supplementary Table S2 available at http://www.BiochemJ.org/bj/424/bj4240179add.htm) showed that the affinity of JSM6427 binding was unaffected by the β subunit partner of huα5; similarly, only small differences were observed between the apparent Ki values of CRRETAWAC for huα5β1-1, huα5β1-2 and huα5huβ1. Similar Ki values were obtained when JSM6427 or CRRETAWAC were used to inhibit binding of the huα5 integrins to 70K (see Supplementary Table S2). These findings show that the α subunit, and not the β subunit, determines the specificity and affinity of antagonist binding. As zfα5 integrins do not recognize human fibronectin or huα5 antagonists, these results suggested that a gain-of-function approach could be employed to define the regions within the α5 subunit that confer ligand-binding specificity.
Swapping blades 1–4 of the zebrafish α5 subunit β-propeller domain results in a complete gain-of-function for binding to human fibronectin and human α5β1 antagonists
To identify regions of the huα5 subunit that are essential for ligand recognition, portions of the zfα5 subunit were exchanged with the corresponding regions of the human subunit (see Supplementary Figure S6) and the binding of fibronectin fragments or antagonists tested in solid-phase assays. Previously, we have used a similar approach to show that a region of the huα5 subunit β-propeller, encompassing blades 2 and 3, contains key sites involved in ligand recognition . As a first step, we swapped blades 2 and 3 of the zfα5 β-propeller with the huα5 sequence and co-expressed the chimaeric α5 subunit with the β1-1–Fc subunit. We found that this exchange was not sufficient to create a receptor that bound to human fibronectin (results not shown); however, exchange of a larger section of the propeller (blades 1–3; W1–W3) showed a weak gain of 50K binding and exchange of blades 1–4 (W1–W4) resulted in a near-complete gain of binding (Figure 4A). Exchange of blades 2–4 (W2–W4) only was not sufficient for full gain of binding, suggesting that all the first four blades are important in forming the ligand-binding pocket for fibronectin. The W1–W4 chimaera recognized the 50K human and 70K zebrafish fibronectin fragments with very similar apparent affinities to those of the wild-type huα5β1-1–Fc (Figure 4, Table 1). The W1–W4 chimaera also expressed the epitopes of human anti-α5 monoclonal antibodies to a similar extent as huα5β1-1 (see Supplementary Figure S10 available at http://www.BiochemJ.org/bj/424/bj4240179add.htm).
We next tested whether the zfα5(huW1–W4)β1-1–Fc chimaeric integrin could recognize the huα5 antagonists. The results (Table 2) showed that the chimaera bound these antagonists with essentially the same apparent Ki values as the wild-type huα5β1-1. Hence, blades 1–4 of the β-propeller contain all the sequences necessary for fibronectin and antagonist binding.
Swapping a loop region in blade 3 of the β-propeller confers a partial gain of function for binding of human α5β1 antagonists
Although swapping a large region of the β-propeller was necessary for gain of fibronectin binding we reasoned that, as the binding interface of low-molecular-mass antagonists is likely to cover a much reduced area compared with that of fibronectin, it may be possible to swap only small regions of the β-propeller to gain antagonist binding. We chose to swap predicted loop regions on the top face of the β-propeller in repeats 2–4, as we have shown previously that these loops are involved in the binding of the RGD motif and CRRETAWAC [28–30]. Three loop-swap mutations were made, KEDTPH to EKEPL (B2–C2 loop), RGRK to SWAA (D3–A3 loop) and RKEIRF to ASSIY (D4–A4 loop) (see Supplementary Figure S6). No changes were made to the B3–C3 loop, as it is identical in both zebrafish and human α5. These mutations only had a small effect on the affinity of 70K binding (Figure 5A and Table 1). We then examined whether JSM6427 or CRRETAWAC could inhibit 70K binding. The results (Figures 5B and 5C) showed that the SWAA loop-swap mutant was strongly inhibited by the antagonists, whereas these compounds had little or no effect on the EKEPL or ASSIY loop-swap mutants. Hence, the D3–A3 loop contains residues that are essential for antagonist recognition. To determine which of these four residues were critical for antagonist binding, we made mutants in which one (RGRA), two (RGAA) or three (SGAA) residues were swapped. JSM6427 inhibited the binding of 70K to the SGAA and RGAA mutants but not the RGRA mutant, and more strongly inhibited 70K binding to the SWAA mutant (Figure 6A). In contrast, CRRETAWAC only inhibited the interaction of 70K with the SWAA mutant (Figure 6B). These results indicate that both the tryptophan residue and the first alanine residue in the SWAA sequence play important roles in JSM6427 binding, but that only the tryptophan residue is critical for gain of CRRETAWAC binding. The serine and the second alanine residues do not appear to be necessary for the gain of JSM6427 binding.
Apparent Ki values for the SWAA mutant (Table 2) showed that the apparent affinity of CRRETAWAC-binding was approx. 3-fold lower, and that for JSM6427 was approx. 10-fold lower, than for wild-type huα5 integrin. Hence, although the D3–A3 loop contains residues that are essential for antagonist recognition, other residues outside of this loop may be necessary for a full gain of function.
A triple-loop-swap results in a complete gain-of-function for binding of human α5β1 antagonists but only a minor gain of binding to human fibronectin
To investigate whether residues other than those in the SWAA sequence are involved in the binding of the antagonists, a construct with a triple-loop-swap (KEDTPH to EKEPL, RGRK to SWAA and RKEIRF to ASSIY; see Supplementary Figure S6) was created. This triple-loop-swap construct only had a minor effect on the affinity of 70K binding (Table 1). We then examined the effects of JSM6427 and CRRETAWAC on 70K binding to this mutant. The results (see Supplementary Figures S11A and S11B available at http://www.BiochemJ.org/bj/424/bj4240179add.htm) showed that 70K binding to the triple-swap mutant was inhibited more potently by the antagonists than the single SWAA swap. Apparent Ki values for the triple-swap mutant were very close to those of wild-type huα5β1-1 integrin (Table 2). Hence, the exchanged residues in the B2–C2 and D4–A4 loops also appear to play a role in antagonist binding.
Although the triple-loop-swap mutant fully restored antagonist binding, this receptor demonstrated only a small increase of binding to 50K (see Supplementary Figure S11C). Hence, the triple swap was not sufficient to restore recognition of human fibronectin.
A new homology model of the α5β1 ligand-binding pocket supports a prominent role for D3–A3 loop residues in binding of human α5 antagonists
Homology modelling of human α5β1 has guided the development of antagonists [19,24,25]. However, these models were based on the medium-resolution αVβ3–cilengitide structure . The αIIbβ3–fibrinogen γ-chain peptide structure, PDB entry 2VDR , was chosen as a template for homology modelling of α5β1 rather than the αVβ3–cilengitide structure (PDB entry 1L5G) for three reasons: (i) the 2VDR structure is of higher resolution than the 1L5G structure (2.4 compared with 3.2 Å); (ii) the 2VDR structure is a high-affinity open conformation of the integrin, whereas the 1L5G structure represents a lower–affinity closed conformation; and (iii) the αV subunit has an aspartate residue in the D3–A3 loop (Asp150) that interacts with the arginyl side chain of the ligand and there is unlikely to be an equivalent interaction in α5 (instead the D3–A3 loop of α5 contains mainly hydrophobic residues and is more similar to αIIb in the same region). However, we used the 1L5G structure for modelling the D4–A4 loop of α5 because the αV subunit has an aspartate residue at the same position as Asp227 of α5, whereas αIIb has a phenylalanine residue. The final model (Figure 7A and see Supplementary Figure S12 available at http://www.BiochemJ.org/bj/424/bj4240179add.htm) shows residues that have been proposed to be critical for ligand binding in α5 ([31,42] and the present study) or β1 [22,24]. On the α subunit side, residues in the D3–A3 and D4–A4 loops (Trp157, Ala158 and Asp227) form walls on either side of the binding pocket for antagonists, whereas Phe187 in the B3–C3 loop lines the base of the pocket. Potentially, other residues in these loops, and the nearby B2–C2 loop, could also participate in antagonist binding. On the β subunit side, residues in the β1-α1 loop of the βI domain (Ser132 and Tyr133) form another wall of the pocket and nearby residues Leu225, Ser227 and Ser172 are also predicted to have essential roles [22,24]. Recognition of the synergy sequence in fibronectin involves residues Tyr208 and Ile210, situated in the C3–D4 loop on the side of the β-propeller . A possible binding mode for the guanidine-mimetic region of JSM6427 is shown in Figure 7(B).
The major findings of the present study are the following: (i) that zfα5 can form heterodimers with β1-1, β1-2 or huβ1 but these receptors do not recognize human fibronectin or human α5β1 antagonists; (ii) swapping the first four blades of the zfα5 β-propeller domain with those of huα5 endows the chimaeric receptor with the ligand-binding properties of huα5β1; (iii) exchange of the D3–A3 loop region in zfα5 with the corresponding region of huα5 (residues Ser156–Ala159) confers zfα5 integrins with the capacity to recognize human α5β1 antagonists; (iv) residues Trp157 and Ala158 are important for the binding of small-molecule antagonists; (v) swapping of three loop regions (the B2–C2, D3–A3 and D4–A4 loops) is sufficient for complete gain of antagonist binding, but not of fibronectin binding; and (vi) homology modelling of the α5β1 ligand-binding pocket supports a prominent role for residues in the D3–A3 loop in binding of human α5β1-specific antagonists.
We found that the zfα5 subunit could form heterodimers with huβ1 in addition to the two zebrafish β integrin subunits. Similarly, huα5 could form heterodimers with β1-1 and β1-2 as well as the huβ1 subunit. Formation of heterodimers is known to be dependent upon an association of the β subunit I domain with the upper face of the α subunit β-propeller. One of the most important interactions that promotes heterodimer assembly is the binding of an arginine/lysine residue in the βD–βD′ loop of the βI domain to a hydrophobic ‘cage’ at the centre of the β-propeller [22,44,45]. Significantly, the aromatic residues that form the cage for the arginine/lysine side chain are conserved between human and zebrafish α5. In addition, the sequence of the βD-βD′ loop is almost identical in β1-1, β1-2 and huβ1. These features may explain why it is possible to form cross-species heterodimers despite the very long evolutionary separation of humans and fish (approx. 350 million years). The high level of identity between the I/A domains of β1-1, β1-2 and huβ1  may also explain why the specificity and affinity of ligand binding was influenced mainly by the α5 subunit and not by the β subunit.
We do not currently have a clear explanation of why zfα5 integrins are unable to interact with human fibronectin. However, an intriguing feature of FN-1 is that a highly conserved asparagine residue in the putative integrin-binding face of the molecule  is replaced with a much less bulky glycine residue (see Supplementary Figures S13). Hence it is possible that steric clashes may interfere with human fibronectin binding to zfα5β1. We found that it was necessary to swap a large section of the β-propeller (repeats 1–4) in order to gain binding to human fibronectin, suggesting that the fibronectin-binding site involves a broad interface. However, it is also possible that a relatively small interface is involved in ligand recognition but that the tertiary structure of the ligand-binding loops is dependent upon the folding of adjacent loops throughout blades 1–4. The key residues involved in fibronectin recognition include Phe187, Tyr208, Ile210 and Asp227 in blades 3 and 4 of the huα5 subunit [42,33,21], and further studies will be necessary to pinpoint additional essential residues. Previously, it has been shown that the fibrinogen-binding site on αIIb involves many residues in blades 2–4, covering a large portion of the upper face and side of the β-propeller . Nearly all these residues are now known to lie in a specialized sub-region of the β-propeller known as the ‘cap’ .
Although zfα5 integrins were unable to bind to human fibronectin, alanine mutagenesis suggested that the same residues involved in the interaction of human fibronectin with the human receptor also participate in zfα5–FN-1 binding. For example, the F183A and D224A mutations of the putative RGD motif-binding site fully abrogated ligand recognition. In addition, we found that mutation of Tyr204 and Leu206 in the C3–D4 loop, equivalent to Tyr208 and Ile210 in huα5, strongly perturbed ligand recognition. Mutation of either Tyr204 or Leu206 to alanine reduced the apparent affinity of fibronectin binding approx. 30-fold, whereas mutation of the corresponding residues in huα5 reduced the affinity of fibronectin binding 5- and 200-fold respectively . These results are consistent with previous reports that loss of interaction with the synergy sequence leads to an appox. 100-fold lower affinity of fibronectin binding [5,6]. A model of the interaction between α5β1 and fibronectin based on X-ray scattering data  predicts that the tyrosine and isoleucine/leucine residues on the side of the β-propeller are located 30–40 Å away from the RGD motif-binding site, which is consistent with the spacing of the synergy sequence from RGD in fibronectin . In agreement with our in vitro results, mutation of Tyr204 to asparagine results in a loss-of-function mutation of α5 in zebrafish . The C3–D4 loop of αIIb contains residues essential for fibrinogen binding by αIIbβ3 , and alternative splicing of this loop can regulate the specificity of ligand interactions in the α7 and αPS subunits [47–49]. In addition, this loop contains the binding site of function-blocking monoclonal antibodies in the αIIb and α5 subunits [22,50]. Hence, the C3–D4 loop may form an important secondary site of ligand recognition in many integrins.
Peptides containing the sequence RGDW or RGDF are potent inhibitors of β3 integrins. The crystal structure of αIIbβ3 bound to the cyclic hexapeptide eptifibatide shows that the GDW sequence has a tight reverse turn following the aspartate residue, and that this bend allows the tryptophan side chain to occupy a hydrophobic pocket formed by Phe160 and Tyr190 on the αIIb subunit . In contrast, RGDW and RGDF peptides are poor inhibitors of α5β1 but peptides containing the sequences RGDGW or RGDGF are very good α5β1 inhibitors [26,27]. The presence of the glycine residue immediately following the aspartate residue (underlined) may favour a more open (less tight) turn in these peptides when binding to α5β1. As noted previously [24,25], replacement of bulky β3 residues Tyr166, Arg214 and Arg216 by Ser177, Gly223 and Leu225 respectively in β1 opens up more space adjacent to the RGD motif-binding pocket. This additional space may allow for the more extended turn in RGDGW/F peptides. In previous homology models of α5β1 [24,25,31], which used the αVβ3 structure  as the template, the side chain of Trp157 was pointing away from the pocket. In our new model, Trp157 and Phe187 of α5 occupy similar positions to Phe160 and Tyr190 in αIIb . The side chains of Trp157 and Phe187 are suitably positioned to allow stacking interactions with the aromatic moieties of peptide antagonists, such as the phenyl group of phenylalanine in RGDGF peptides and the indole ring of tryptophan in RGDGW and RRETAWA peptides. Hence, our model helps to explain why these moieties are critical for high-affinity binding of these antagonists.
We found that the specificity of antagonist binding appears to derive mainly from the α subunit. In our model (Figure 7A), residues in huβ1 predicted to be important for ligand recognition by α5β1  are completely conserved in zebrafish β1-1 and β1-2 . Ligand-binding pocket residues in huα5 are also conserved in zfα5, with the important exceptions that Trp157 is substituted by glycine and Ala158 by arginine. We have previously shown that Trp157 in the D3–A3 loop is involved in binding of human α5β1-specific and α5β1-selective antagonists such as CRRETAWAC and RGDGW [28–30]. In the present study we have demonstrated that residues in this loop (SWAA) are also important for the binding of small molecule antagonists. Swapping only two loop residues (the RGAA mutant) gave a partial gain-of-function for binding of JSM6427 but no gain of binding for CRRETAWAC. Hence, although the tryptophan residue is essential for high-affinity binding of CRRETAWAC, this residue plays a more minor role in the interaction of α5β1 with JSM6427. Instead, our results suggest that Ala158 has an important function in the binding of this compound, either by direct interaction or by creating space for other interactions. The 4-methoxy substituent of the 2-aminopyridine ring of JSM6427 is involved in high-affinity binding to α5β1 , and our modelling (Figure 7B) suggests that this moiety could interact with residues in the D3–A3 loop region.
JSM6427 is undergoing Phase I clinical trials for treatment of age-related macular degeneration [51, 52]. Our findings will aid in the rational design of more potent and specific α5β1 antagonists. Finally, animal models such as zebrafish provide a valuable means of drug screening . However, owing to sequence differences in the D3–A3 loop, antagonists of human integrins often do not cross-react with integrins of other species ([54,55] and the present study). Our results suggest that introduction of the SWAA loop swap into the zfα5 gene could create an in vivo model suitable for drug screening or discovery .
Paul Mould and Ewa Koper performed the experiments. Paul Mould designed the project and wrote the manuscript. Paul Mould and Adam Byron carried out the molecular modelling and docking. Grit Zahn and Martin Humphries helped with experimental design and manuscript preparation/revision.
This work was supported by The Wellcome Trust [grant numbers 045225, 074941]. G. Z. is an employee of Jerini AG.
We thank Dr Adam Hurlstone, Dr Scott Holley and Dr Jordi Bella for helpful discussions, Simon Williams, Patrick Buckley and Guillaume Jacquemet for assistance with homology modelling. We also thank Scott Holley for the gift of oligonucleotides, Sumito Koshida for zebrafish α5 cDNA, John Wilkins and Kenneth Yamada for antibodies and Janet Askari for human α5 and β1–Fc constructs. We are grateful to Elizabeth Lord, Jennifer Hamilton and Simon Mathers for technical assistance, and Amanda Kelly and Paul Walker for PAC2 cell line and cDNA. We thank Roland Stragies (Jerini AG) for synthesis of JSM6427 and JSM6406, and Ulf Reimer (Jerini AG) and Patrick Bryant for helpful discussion of homology modelling.
Abbreviations: 50K, 50 kDa cell-binding domain of human fibronectin; 70K, 70 kDa cell-binding fragment of zebrafish FN-1; CHO, Chinese-hamster ovary; DMEM, Dulbecco's modified Eagle's medium; FN-1, zebrafish fibronectin-1; hu, human integrin subunit; MIDAS, metal-ion-dependent adhesion site; W, β-propeller blade; zf, zebrafish integrin subunit
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