1. Field of the Invention
The invention in the field of structural biology and biochemistry relates to a novel 3D structure determined by x-ray crystallography of a ternary complex of the amino terminal fragment (ATF) of the urokinase-type plasminogen activator (uPA) together with a soluble form of its cell surface receptor (suPAR) and an antibody that binds to suPAR without disrupting ATF-suPAR binding, as well as uses of this structural information to design or screen putative inhibitors of ATF-suPAR interactions. The invention also relates to novel methodologies to generating binary, ternary or quartenary complexes of suPAR, ATF-suPAR, uPA-suPAR with ligands such as an antibody against suPAR or against uPA, or other ligands for suPAR such as integrins and vitronectin for the purpose of generating crystals that diffract to high resolution and are therefore expected to yield high resolution structures suitable for drug discovery and structure based drug design.
2. Description of the Background Art
Urokinase-type plasminogen activator (uPA) together with its cell surface receptor (uPAR) mediate surface-bound plasminogen activation (Myohanen, H et al. (2004) Cell Mol Life Sci 61:2840-58), and have been recognized to play important roles in a variety of cellular functions, including cell adhesion, migration, tissue remodeling, and tumor invasion (Andreasen, P A et al. (2000) Cell Mol Life Sci 57:25-40; Blasi, F et al. (2002) Nat Rev Mol Cell Biol 3:932-43; Ploug, M (2003) Curr Pharm Des 9:1499-528; Mondino, A et al. (2004) Trends Immunol 25:450-5). The molecular basis of these broad physiological roles comes from uPAR's capability to interact with many ligands, e.g., uPA, vitronectin, β1-, β2- and β3-integrins, G-protein coupled receptors, etc. Knowledge of the three-dimensional (3D) structure of uPA/uPAR complexes will provide crucial insights into the molecular mechanisms responsible for many of the unique properties of the uPA-uPAR interaction.
uPA is made up of a serine protease domain located at its carboxy-terminus (C-terminus) and a modular amino-terminal (N-terminal) fragment “ATF”, amino acid residues 1-135 (also referred to herein as uPA1-143) that includes a growth factor-like domain (GFD) and a kringle domain (KrD). uPA1-143 of uPA is responsible for the receptor binding, forming a stable complex with a dissociation constant of 0.28 nM (Ploug, M et al. (1994) FEBS Lett 349:163-8). uPAR is a 313-amino acid glycoprotein linked to the cell surface through a C-terminal glycosyl phosphatidylinositol (GPI) anchor (Ploug et al., supra). Soluble uPAR variants (suPAR) consisting of residues uPAR1-277 without the GPI anchor have been identified under physiologic and pathological conditions, such as in patients with malignancies (Pappot, H et al (1997) Eur J Cancer 33:867-72) or paroxysmal nocturnal hemoglobinuria (PNH) (Ronne, E et al (1995) Br J Haematol 89:576-81; Gao, W et al (2002) Int J Hematol 75:434-9). suPAR binds uPA with a Kd in the subnanomolar range that is indistinguishable from the GPI-anchored full-length uPAR (Ploug et al., supra), indicating that suPAR is an appropriate candidate for the structural study of uPA-uPAR interactions in vitro.
The present inventors disclose the crystal structure at a 1.9 Å resolution of suPAR-uPA1-143 further complexed with a Fab fragment of mAb, ATN-615, that was raised against suPAR. The ternary complex is referred to as “uPAR-uPA1-143-Fab” or as “uPAR/uPA1143/Fab”.
Based on knowledge from this structure, the present invention permits design and/or testing of more universal antagonists of uPA-uPAR interactions not limited by species specificities. This derives from the finding that amino acid residues involved in the first region of the uPAR-uPA interface are highly conserved among different species, so that an antagonist that targets this region inhibits human and mouse (and rat, etc.) uPAR-uPA interactions.
The present invention provides a platform for rational design of inhibitors of uPAR-uPA interactions that would be expected to prevent, reverse or attenuate the pathophysiological consequences of these interactions. One example of such consequences is tumor metastasis.
The present invention also relates to methods for forming crystals that diffract to high resolution. In the absence of an antibody that binds to the ATF-suPAR complex, crystals of ATF-suPAR diffract much more poorly (to 3.1 Å). The present invention describes the use of ligands for uPA or uPAR including antibodies, peptides, other proteins and small molecules that, when bound, allow the formation of crystals that diffract to high resolution.
In a preferred embodiment, the present invention is directed to a composition comprising a crystallized complex of uPA or a fragment thereof bound to a soluble uPAR molecule and further bound to, and constrained by, a ligand that has an affinity for uPAR of at least about 100 μM, preferably at least about 1 μM, more preferably at least about 10 nm, and which binds uPAR without disrupting uPA-uPAR binding interactions.
In the above composition, the uPA is preferably human uPA and the uPAR is preferably human uPAR and the ligand is preferably a uPAR-specific antibody or antigen-binding fragment thereof. Alternatively, the ligand may be a uPA-specific antibody or an antigen-binding fragment thereof. A preferred antibody is the anti-uPAR mAb designated ATN-615.
The above composition preferably is characterized by having a 3D atomic structure of the complex defined by a set of structural coordinates corresponding to the set of structural coordinates set forth in Table 1 and
Also provided is a computing platform for generating a 3D model of a uPA-uPAR complex further constrained by a uPAR ligand, which computing platform comprises:
A computer generated model of the present invention preferably represents the conformationally constrained 3D structure of a uPA-uPAR complex to which is also bound a ligand for uPAR, the computer generated model having a 3D atomic structure defined by a set of x-ray crystallographic coordinates set out in Table 1 and
The invention includes a computer readable medium comprising, in a retrievable format, data that include a set of structure coordinates defining at least a portion of a 3D crystallographic structure of a crystallized uPA-uPAR complex that is conformationally constrained by being bound to a ligand for uPAR. A preferred ligand above is an Fab fragment of mAb ATN-615
In the above computer readable medium, the structure coordinates defining at least a portion of a 3D structure of the crystallized complex correspond to a set of coordinates set forth in Table 1 and
This invention includes a method of crystallizing a ternary complex of uPA, soluble uPAR and a ligand for uPAR:
The present inventors conceived of adding a uPAR binding partner, such as a uPAR-specific antibody or fragment (e.g., an Fab fragment) to constrain the 3D structure and thereby facilitate crystallization and improve the diffraction resolution of uPAR-uPA1-143 complexes. The mAb ATN-615, which had been raised by some of the present inventors against suPAR and which binds to suPAR at a domain so that such binding does not disrupt uPA-uPAR interactions was used in this capacity and is exemplified herein. Indeed, the ATN-615 Fab fragment facilitated suPAR-uPA1-143 crystallization, greatly improved the diffraction resolution of the crystals to 1.9 Å and provided phasing power to generate a discernible electron density map for suPAR and uPA1-143 model building.
Antibodies are not the only type of uPAR binding partners that may be used in the present invention. Any ligand for uPAR that binds to uPAR without disrupting the binding of uPA to uPAR or altering the structure of the uPA-uPAR complex may be used for similar structural analysis. Examples of suPAR binding partners are vitronectin (Vn) and various integrins. Similarly, any binding partner that binds to uPA without altering its structure or interfering with its binding to uPAR may also be used for structural analysis. Other ligands useful as above include peptides, phages, small organic molecules, aptamers, and the like that bind either to suPAR or uPA.
By enabling a structural determination of the uPA-uPAR binding interaction at a new level of resolution, the present invention enables the testing and screening of potential inhibitors or antagonists of this interaction. First, the restrictive species specificity of uPA-uPAR interactions is an impediment to testing inhibitors in murine or other rodent systems, for example. The present structure shows that residues involved in the first region of the uPAR-uPA interface are highly conserved among different species so that antagonists targeting this region would inhibit both human and mouse uPAR-uPA interactions.
The structure of uPAR-uPA1-143 complex described herein serves as a platform for rational design of inhibitors of uPAR-uPA interactions.
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.
uPAR and uPA1-143 (amino acid residue 1-143 uPA) were expressed in drosophila S2 cells and purified as described (Huang, M, A P Mazar, et al (2005) Acta Crystallogr D Biol Crystallogr 61(Pt 6):697-700). The suPAR-uPA1-143 complex was formed by incubating uPA1-143 with suPAR at a 1:1 molar ratio at room temperature in 50 mM HEPES and 100 mM NaCl pH 7.4 and was purified on a Superdex75 gel filtration column. The suPAR-uPA1-143 complex was then mixed at 1:1 molar ratio with Fab fragment of anti-suPAR antibody, ATN-615, and the mixture was purified on a superdex 200 column. The 1:1:1 suPAR-uPA1-143 Fab was concentrated to 10 mg/ml using Millipore Ultrafree centrifugal filters.
Diffracting quality crystals of the suPAR-uPA1-143-ATN-615 ternary complex were generated by microdialysis (McPherson, A., Preparation and Analysis of Protein Crystals, John Wiley & Sons, 1992, pp 88-91) with 4% PEG4K, 5% ethylene glycol, 5% methanol, 0.05% sodium azide, 50 mM cacodylate pH 6.5. The crystals typically appeared in 3 to 7 days, and grew to a maximal size of 0.03×0.05×0.1 mm3. The crystals are harvested from dialysis button, and brief soaked in a cryoprotectant of 20% glycerol, 20% PEG4K, 5% methanol, 50 mM cacodylate pH 6.5.
A complete data set of the ternary complex to 1.9 Å was collected using synchrotron radiation at the Advanced Photon Source (APS), Argonne National Laboratory. See
The resulting model was refined using CNS and manual model fitting was carried out using the program O. The final model consists of 427 ATN residues (L and H chains), 122 uPA1-143 residues (A chain), 249 suPAR residues (U chain), 3 N-acetylglycosamines (V chain); 21 disulfide bonds; 1 glucose (V chain), 335 waters (W chain), 1 SO4 (S chain) and 7 (poly)-ethylene glycol moieties (P chain).
uPAR dimerizes in detergent-resistant lipid rafts on cell surfaces (Cunningham, O et al (2003) EMBO J 22:5994-6003). Recombinant suPAR from Drosophila S2 cells also tends to form oligomers in aqueous solution at concentrations required for protein crystallization (Llinas et al., supra). This posed great difficulties in trying to study uPAR's crystal structure. Previous studies by various of the present inventors and others showed that uPA could regulate uPAR oligomerization in vivo at the cellular level (Sidenius, N. et al (2002) J Biol Chem 277:27982-90) and dissociate suPAR oligomers in vitro (Shliom, O. et al (2000) J Biol Chem 275:24304-12), leading to the formation of crystallizable uPAR-uPA1-143 complexes at a 1:1 ratio. However, crystals obtained from this complex diffracted to only 3.1 Å (Huang et al., supra.)
The present inventors thus conceived of adding a uPAR binding partner, such as a uPAR-specific antibody or fragment (e.g., an Fab fragment) to constrain the 3D structure and thereby facilitate crystallization and improve the diffraction resolution of uPAR-uPA1-143 complexes. The mAb ATN-615, which had been raised against suPAR and which binds to suPAR at a domain that does not disrupt uPA-uPAR interactions was used in this capacity and is exemplified herein. Indeed, the ATN-615 Fab fragment facilitated suPAR-uPA1-143 crystallization, greatly improved the diffraction resolution of the crystals to 1.9 Å and provided phasing power to generate a discernible electron density map for suPAR and uPA1-143 model building.
The electron density map disclosed herein shows that the majority of the structure in the uPAR-uPA1-143-Fab complexes was well-ordered. The receptor binding region of uPA is clearly defined in the electron density map (
Because uPA1-143 binds mainly to the D1 domain of uPAR, and ATN-615 recognizes only the D3 domain at the other side, the three proteins in the ternary complex arrange into a linear and elongated complex with a length of 141 Å (
Data collection and refinement statistics are summarized in Table 1 below and the x-ray crystallographic details and coordinates appear in
The structure of the ternary complex as analyzed by the present X-ray analysis reveals both the uPA7-43 (GFD) and the uPA50-135 (KrD) domains of uPA1-143 (cyan-colored molecule in
In the unbound state, a structure obtained using NMR shows that the two domains of uPA1-143 and the KrD, exhibit a high degree of structural independence involving little or no inter-domain interaction (
The structure of suPAR consists of 17 antiparallel β strands with three short α-helices, which are organized into three domains (
The D1 domain comprises residues uPAR1-80 and a six-stranded antiparallel continued β-sheets (β1 to β6) constrained by three disulfide bonds. The β5 strand (uPAR53-58) is highly conserved across species and is essential for D1-D2 association. The D2 residues (uPAR92-191) form a β sheet with six strands (β7 to β12), a short α-helix (α1, uPAR104-107) between β7 and β8, and four disulfide bonds. An interesting feature of D2 is that the β10 strand (uPAR143-149) twists about 60° at Gly146, so that the N-terminal half of this strand (uPAR143-145) is parallel with D2 β9, whereas the C-terminal half (uPAR147-149) lines up with the β5 of another domain (D1), suggesting a role in linking the domains (
Structural superposition of the current uPAR structure with the suPAR in complex with a peptidyl inhibitor (Llinas et al., supra) shows that each domain of these two structures share similar folding with relatively small root-mean-squared deviation (rmsd) between two the structures, namely, 1.5 Å for D1 (77 Cα), 2.2 Å for D2 (89 Cα), 1.3 Å for D3 (81 Cα), 2.4 Å for D1D2 (166 Cα), 4 Å for D2D3 (170 Cα), respectively. However, the relative domain positions in the two suPAR structures show dramatic differences (
Compared with the presently described suPAR structure, the three loops (uPAR16-23, uPAR46-53 and uPAR149-156) in the uPAR-inhibitor complex of Llinas et al., are shifted away from the center of binding cavity by about 13 Å, 8.5 Å and 5.6 Å, respectively, and six β strands in D1 domain shift by about 5-10 Å in order to enlarge the bottom of the binding cavity to accommodate the peptidyl inhibitor. Two loops (uPAR99-104) and (uPAR128-143) located at the opening of the uPAR cavity also show significant changes. Loop uPAR99-104 moves closer to D1 by 5.2 Å upon peptide (inhibitor) binding and this movement creates more space for uPA1-143 binding. Parts of the loop uPAR128-143 are disordered in both structures, but the stretch at both ends (residues 130-128 and 139-143) shows that this loop may play an important role in uPA1-143 binding. The domain associations D1-D2 and D1-D3 also undergo significant changes between two structures. In the suPAR/peptidyl complex, the D1-D3 interface decreases in area to 169 Å2 and no hydrogen bond interaction were observed in this interface; the D1-D2 domain interface, especially β7, β8, β10 and the loop uPAR100-104 of D2 and β5 of D1 domain, also undergo significant shifts. These results highlight the dramatic conformational changes induced in uPAR by the binding of the peptidyl inhibitor and indicate that the domain-domain associations and the loops linking β-strands in uPAR are quite flexible. This suggests caution in designing uPAR inhibitors.
uPA1-143 inserts into the cavity of uPAR (in a fashion that may be viewed as analogous to a teaspoon sitting in a teacup) forming a large interface of 1171 Å2 (
The second region of the uPAR-uPA interface is localized at the D1 hydrophobic patch (shown above), which interacts with three hydrophobic residues of uPA-F25, I128 and W30 (
The third region is localized to the edge of the teacup-shaped cavity and consists of a hydrogen bond (Q40 of uPA and T8 of uPAR D1) and van der Waals forces between uPAR and residues (Q40 and H87 from KrD) of uPA.
These results indicate that the uPAR D1 and D2 domains play important roles in the binding uPA. However, the D3 domain also undergoes direct interactions with uPA. Part of D3 (α3, uPAR253-255) contacts uPA by van der Waals interactions. D3 also forms a wall of the uPAR cavity and maintain the closeness of the cavity by interacting with D1 (
uPA-uPAR binding is strongly species specific, as least between human and mouse (Ploug, M, S Ostergaard, et al (2001) Biochemistry 40:12157-68). Little or no binding occurs between human uPAR and murine uPA, and vice versa (Estreicher, A et al. (1989) J Biol Chem 264:1180-9.
Sequence alignment of uPAR residues involved in uPA binding show that most hydrophobic residues (4 of 5, that is, V29, L31, L40, L55, L66) and charged residues (5 out of 6, T8, R53, E68, T127, D140, H166) are conserved in all different species of uPAR. The only significant difference in murine uPAR compared to human uPAR is a change from L to E at residue 31. In human uPAR, L31 is a part of the hydrophobic patch (
On the uPA side, sequence alignment of the binding residues of uPA1-143 (or uPA1-135) in different species (Table 2) indicates significant variation of receptor binding residues (underscored in Table 2) in mouse when compared with human uPA. The W30R replacement in murine (vs. human) uPA is notable because human W30 is part of a hydrophobic cluster interacting with human uPAR's hydrophobic patch. Humanization of the murine uPA7-43 by an R30W mutation (along with other mutations such as Y22N) resulted in high-affinity ligand for human uPAR (Quax, P H, J M Grimbergen, et al (1998) Arterioscler Thromb Vasc Biol 18:693-701.
This species specificity makes it difficult to test or screen for potential inhibitors of human uPAR-uPA interaction using mouse or rat uPAR (Ploug, 2003, supra; Behrendt, N (2004) Biol Chem 385:103-36). The structure defined herein provides a potential solution for this problem. Because residues involved in the first region of the uPAR-uPA interface are highly conserved among different species, antagonists targeting this region should inhibit both human and mouse uPAR-uPA interactions.
The structure of uPAR-uPA1-143 complex described herein provides a model that unifies and validates a large body of biochemical research on uPAR-uPA interactions (Ploug, 2003, supra; Behrendt, supra). Moreover, it provides a platform for rational design of inhibitors of uPAR-uPA interactions that may prevent, reverse or attenuate the pathophysiological consequences of these interactions, as in tumor metastasis.
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The references cited herein are all incorporated by reference herein, whether specifically incorporated or not.
Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.
This invention was funded in part by a grant RO1 HL60169 from the National Heart Lung and Blood Institute of the National Institutes of Health (to co-inventor D. Cines) which provides to the United States government certain rights in this invention.
Filing Document | Filing Date | Country | Kind | 371c Date |
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PCT/US2006/038608 | 10/3/2006 | WO | 00 | 11/26/2008 |
Number | Date | Country | |
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60722402 | Oct 2005 | US |