Discovery of a novel conformational equilibrium in urokinase-type plasminogen activator

Although trypsin-like serine proteases have flexible surface-exposed loops and are known to adopt higher and lower activity conformations, structural determinants for the different conformations have remained largely obscure. The trypsin-like serine protease, urokinase-type plasminogen activator (uPA), is central in tissue remodeling processes and also strongly implicated in tumor metastasis. We solved five X-ray crystal structures of murine uPA (muPA) in the absence and presence of allosteric molecules and/or substrate-like molecules. The structure of unbound muPA revealed an unsuspected non-chymotrypsin-like protease conformation in which two β-strands in the core of the protease domain undergoes a major antiparallel-to-parallel conformational transition. We next isolated two anti-muPA nanobodies; an active-site binding nanobody and an allosteric nanobody. Crystal structures of the muPA:nanobody complexes and hydrogen-deuterium exchange mass spectrometry revealed molecular insights about molecular factors controlling the antiparallel-to-parallel equilibrium in muPA. Together with muPA activity assays, the data provide valuable insights into regulatory mechanisms and conformational flexibility of uPA and trypsin-like serine proteases in general.

Two additional washes were performed with 0.25 % (v/v) and 0 % (v/v) Triton X-100 respectively. Finally the inclusion bodies was resuspended in denaturation buffer (50 mM Tris pH 8.0; 100 mM NaCl; 10 mM β-mercaptoethanol; 6 M urea; 1 mM EDTA) and denatured by slow stirring at 4 °C. Next the protein concentration was adjusted to below 0.2 mg/mL in denaturation buffer and dialysed against 10 L of refolding buffer (50 mM Tris pH 8.0; 1 mM β-mercaptoethanol; 3 M urea; 10 % glycerol) at 4 °C for 22 h. Urea was removed by dialysis against 2x10 L of buffer containing 50 mM Tris pH 8.0 and 10% glycerol at 4 °C for 22 h. After solubilisation and refolding muPA was subsequently captured on nickelsepharose and eluted in 50 mM Bicine pH 8.0; 500 mM NaCl and 400 mM imidazole and dialyzed extensively against PBS (10 mM Na 2 HPO 4 ; 1.8 mM KH 2 PO 4 ; 2.7 mM KCl; 137 mM NaCl; pH 7.4). The protein concentration was adjusted to 0.5 mg/mL and incubated with 2.5 µg/mL plasmin at 22°C for 22h. The incubation with plasmin ensured correct cleavage between Lys15 and Ile16 to generate the catalytic domain. Plasmin was removed by passing the sample over a CNBr (GE Healthcare) activated aprotinin sepharose column. To remove non-activated or non-correctly folded protein benzamidine-sepharose (GE Healthcare) chromatography was applied. Finally the active catalytic domain was purified by sizeexclusion chromatography on a Superdex 75 equilibrated with PBS supplemented with 300 mM NaCl. Protein purity was verified by SDS-PAGE analysis. EGR-cmk-bound muPA was prepared by incubating muPA with 10-fold molar excess of EGR-cmk for 1 h at 22 °C in PBS. Excess EGR-cmk was removed by dialyzing against 2 L PBS at 4 °C for 16 h.
Crystallization. All crystals were grown using the hanging drop vapor diffusion method, with 1:1 (v/v) ratio of protein to reservoir solution. For all proteins initial hits were identified using commercially available screens including Structure Screen 1, Structure Screen 2, JCSG-plus, Clear Strategy Screen 1 and Clear Strategy Screen 2 (Molecular Dimensions). For the catalytic domain of muPA (apo-muPA) final crystals were grown using 20 mg/mL apo-muPA at 18 °C using 4 µL drops equilibrated over 1 mL of 100 mM HEPES, pH 7.4 and 1.8 M Li s SO 4 . For the muPA:Nb22 complex, prior to crystallization experiments, 10 mg/ml of the catalytic domain of muPA was incubated with 2-fold molar excess of Nb22 at 4 °C. The complex was purified on a Superdex 75 and verified by SDS-PAGE analysis.
Final crystals were grown using 6 mg/mL of the muPA:Nb22 complex using 2 µL drops equilibrated over 1 mL of reservoir solution containing 0.2 M Ammonium Acetate, 0.1 M Tris pH 8.0, 16 % (w/v) PEG10,000. For the muPA:Nb7 complex, prior to crystallization experiments, 10 mg/ml of the catalytic domain of muPA was incubated with 2-fold molar excess of Nb7 at 4 °C. The complex was purified on a Superdex 75 and verified by SDS-PAGE analysis. Final crystals were grown using 6 mg/mL of the muPA:Nb7 complex using 4 µL drops equilibrated over 1 mL of reservoir solution containing 100 mM HEPES pH 7.4 and 1.6 M Li 2 SO 4 . Active site occupied muPA were prepared by soaking muPA:Nb7 crystals with 1 mg/mL H-Glu-Gly-Arg-chloromethylketone (EGR-cmk, Bachem) or p-aminobenzamidine (Sigma) for 24 h before harvesting the crystals. In the structure of apo-muPA 8 sulfate ions (2 for each muPA molecule) originating form the crystallization buffer is observed on the surface of muPA, whereas 4 nickle ions (1 for each muPA molecule) likely to originate from the purification procedure occupies solvent channels between two adjacent muPA molecules.
In the structure of muPA:Nb7 we observed 7 sulfate ions (3 on the surface of muPA and 4 on the surface of Nb7) originating from the crystallization buffer, whereas additional sulfate ions occupy binding pockets in the active site region of muPA in the muPA:Nb7:pamiobenzamidine and muPA:Nb7:EGR-cmk structures.
Fluorescent assay. Full-length muPA (0.23 µM) was mixed with paminobenzamidine (60 µM) and incubated for 15 min at 22°C before adding Nb7 (3 µM) or Nb22 (800 nM). An irrelevant control nanobody (800 nM) or the active site binding peptide mupain-1 (10 µM) was used as a negative and positive control respectively. Fluorescence emission spectrums were recorded at 25°C on a PTI quantamaster spectrofluorometer in a 2 mm x 10 mm quarts cuvette. An emission scan of 340 -400nm using an excitation wavelength of 335nm and an integration of 1-2 s over a 1.0 nm step resolution was used. The buffer used was HBS supplemented with 0.1 % (w/v) polyethyleneglycol 8000.
Surface Plasmon Resonance. EGR-cmk inhibited muPA was prepared by incubating muPA with 10-fold molar excess of EGR-cmk for 1 h at 22 °C in PBS. Excess EGR-cmk was removed by dialyzing against 2 L PBS at 4 °C for 16 h. The equilibrium dissociation constant ! , the association rate !" , and the dissociation rate !"" of Nb7 binding to full-length muPA, the catalytic protease domain of muPA and their EGR-cmk active site inhibited variants were determined by surface plasmon resonance on a Biacore T200 (GE Healthcare).
Nb7 was diluted to 0.5 µg/mL in immobilization buffer (10 mM sodium acetate pH 5), and immobilized on a CM5 sensor chip (GE Healthcare) by amine coupling to approximately 100 response units. The muPA variants were diluted in running buffer HBS+0.1% (w/v) BSA and injected for 380s onto the immobilized Nb7 with a flow rate of 30 µL/min. The dissociation was monitored for 600s before regenerating the surface with 10 mM Glycine, 0.5 M NaCl, pH 2.5. The kinetic constants were determined at 22 °C, and the experimental curves were fitted to a 1:1 binding model using the BiaCore evaluation software.