Cai Yuan

Structural basis for recognition of urokinase-type plasminogen activator by plasminogen activator inhibitor-1

Plasminogen activator inhibitor-1 (PAI-1), together with its physiological target urokinase-type plasminogen activator (uPA), plays a pivotal role in fibrinolysis, cell migration, and tissue remodeling and is currently recognized as being among the most extensively validated biological prognostic factors in several cancer types. PAI-1 specifically and rapidly inhibits uPA and tissue-type PA (tPA). Despite extensive structural/functional studies on these two reactions, the underlying structural mechanism has remained unknown due to the technical difficulties of obtaining the relevant structures. Here, we report a strategy to generate a PAI-1·uPA(S195A) Michaelis complex and present its crystal structure at 2.3-Å resolution. In this structure, the PAI-1 reactive center loop serves as a bait to attract uPA onto the top of the PAI-1 molecule. The P4–P3′ residues of the reactive center loop interact extensively with the uPA catalytic site, accounting for about two-thirds of the total contact area. Besides the active site, almost all uPA exosite loops, including the 37-, 60-, 97-, 147-, and 217-loops, are involved in the interaction with PAI-1. The uPA 37-loop makes an extensive interaction with PAI-1 β-sheet B, and the 147-loop directly contacts PAI-1 β-sheet C. Both loops are important for initial Michaelis complex formation. This study lays down a foundation for understanding the specificity of PAI-1 for uPA and tPA and provides a structural basis for further functional studies.

Trp2313-His2315 of factor VIII C2 domain is involved in membrane binding: structure of a complex between the C2 domain and an inhibitor of membrane binding.

Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 Å) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed β-strand of the C2 domain, Trp2313-His2315. This result indicates that the Trp2313-His2315 segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.

A Novel Tumor Targeting Drug Carrier for Optical Imaging and Therapy

Human serum albumin (HSA), a naturally abundant protein in blood plasma and tissue fluids, has an extraordinary ligand-binding capacity and is advocated as a drug carrier to facilitate drug delivery. To render it tumor targeting specificity, we generated a recombinant HSA fused with the amino-terminal fragment (ATF) of urokinase, allowing the fusion protein to bind to urokinase receptor (uPAR), which is shown to have a high expression level in many tumors, but not in normal tissues. To test the efficacy of this bifunctional protein (ATF-HSA), a hydrophobic photosensitizer (mono-substituted β-carboxy phthalocyanine zinc, CPZ) was chosen as a cytotoxic agent. A dilution-incubation-purification (DIP) strategy was developed to load the ATF-HSA with this CPZ, forming a 1:1 molecular complex (ATF-HSA:CPZ). We demonstrated that CPZ was indeed embedded inside ATF-HSA at the fatty acid binding site 1 (FA1) of HSA, giving a hydrodynamic radius of 7.5 nm, close to HSAs (6.5 nm). ATF-HSA:CPZ showed high stability and remarkable optical and photophysical properties in aqueous solution. In addition, the molecular complex ATF-HSA:CPZ can bind to recombinant uPAR in vitro and uPAR on tumor cell surfaces, and was efficient in photodynamic killing of tumor cells. The tumor-killing potency of this molecular complex was further demonstrated in a tumor-bearing mouse model at a dose of 0.080 μmol / kg, or 0.050 mg CPZ / kg of mouse body weight. Using fluorescent molecular tomography (FMT), ATF-HSA:CPZ was shown to accumulate specifically in tumors, and importantly, such tumor retention was higher than that of HSA:CPZ. Together, these results indicate that ATF-HSA:CPZ is not only an efficient tumor-specific cytotoxic agent, but also an useful tumor-specific imaging probe. This bifunctional protein ATF-HSA can also be used as a drug carrier for other types of cytotoxic or imaging agents to render them specificity for uPAR-expressing tumors.

Crystal structure of the urokinase receptor in a ligand-free form.

The urokinase receptor urokinase-type plasminogen activator receptor (uPAR) is a surface receptor capable of not only focalizing urokinase-type plasminogen activator (uPA)-mediated fibrinolysis to the pericellular micro-environment but also promoting cell migration and chemotaxis. Consistent with this multifunctional role, uPAR binds several extracellular ligands, including uPA and vitronectin. Structural studies suggest that uPAR possesses structural flexibility. It is, however, not clear whether this flexibility is an inherent property of the uPAR structure per se or whether it is induced upon ligand binding. The crystal structure of human uPAR in its ligand-free state would clarify this issue, but such information remains unfortunately elusive. We now report the crystal structures of a stabilized, human uPAR (H47C/N259C) in its ligand-free form to 2.4 Å and in complex with amino-terminal fragment (ATF) to 3.2 Å. The structure of uPAR(H47C/N259C) in complex with ATF resembles the wild-type uPAR·ATF complex, demonstrating that these mutations do not perturb the uPA binding properties of uPAR. The present structure of uPAR(H47C/N259C) provides the first structural definition of uPAR in its ligand-free form, which represents one of the biologically active conformations of uPAR as defined by extensive biochemical studies. The domain boundary between uPAR DI-DII domains is more flexible than the DII-DIII domain boundary. Two important structural features are highlighted by the present uPAR structure. First, the DI-DIII domain boundary may face the cell membrane. Second, loop 130-140 of uPAR plays a dynamic role during ligand loading/unloading. Together, these studies provide new insights into uPAR structure-function relationships, emphasizing the importance of the inter-domain dynamics of this modular receptor.

Structure of catalytic domain of Matriptase in complex with Sunflower trypsin inhibitor-1.

BackgroundMatriptase is a type II transmembrane serine protease that is found on the surfaces of epithelial cells and certain cancer cells. Matriptase has been implicated in the degradation of certain extracellular matrix components as well as the activation of various cellular proteins and proteases, including hepatocyte growth factor and urokinase. Sunflower trypsin inhibitor-1 (SFTI-1), a cyclic peptide inhibitor originally isolated from sunflower seeds, exhibits potent inhibitory activity toward matriptase.ResultsWe have engineered and produced recombinant proteins of the matriptase protease domain, and have determined the crystal structures of the protease:SFTI-1 complex at 2.0 Å as well as the protease:benzamidine complex at 1.2 Å. These structures elaborate the structural basis of substrate selectivity of matriptase, and show that the matriptase S1 substrate specificity pocket is larger enough to allow movement of benzamidine inside the S1 pocket. Our study also reveals that SFTI-1 binds to matriptase in a way similar to its binding to trypsin despite the significantly different isoelectric points of the two proteins (5.6 vs. 8.2).ConclusionsThis work helps to define the structural basis of substrate specificity of matriptase and the interactions between the inhibitor and protease. The complex structure also provides a structural template for designing new SFTI-1 derivatives with better potency and selectivity against matriptase and other proteases.

Does the urokinase receptor exist in a latent form

Abstract.Multiple cellular functions of urokinase and its receptor are associated with the receptor’s capability to interact with a number of ligands at the molecular level. The presence of urokinase is generally needed for the urokinase receptor to acquire this capability. Recent X-ray studies of the structure of the urokinase receptor in complex with either its ligand or peptide inhibitors demonstrate the flexibility of the domain organization of the receptor, suggesting that unliganded urokinase receptor may exist in a latent form that has a conformation different from its ligand-binding form.

Crystal Structures of Matriptase in Complex with Its Inhibitor Hepatocyte Growth Factor Activator Inhibitor-1

Background: Matriptase requires very strict regulation by its inhibitor, hepatocyte growth factor activator inhibitor-1. Results: Crystal structures of matriptase serine protease domain and its complex with HAI-1 were determined. Conclusion: These structures elucidate the structural basis of inhibition of matriptase by HAI-1 KD1. Significance: This work provides important structural insights for the future design of small molecular inhibitors. Matriptase, a type II trans-membrane serine protease of the S1 trypsin-like family, is expressed on the surface of nearly all normal human epithelium and found in biological fluid-like human milk. Matriptase overexpression has been implicated in tumor progression in certain epithelium-derived cancer cells. Matriptase is tightly regulated by its cognate inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1). It has been demonstrated that the Kunitz domain I (KD1) but not Kunitz domain II (KD2) of HAI-1 is responsible for the inhibitory activity of HAI-1 against matriptase. To investigate the molecular basis of inhibition of matriptase by HAI-1, we solved several crystal structures of matriptase serine protease domain in complex with the fragments of HAI-1. Based on these structures, we found that the binding of KD1 was different from previously predicted binding mode. The P3 arginine residue occupies the S3 specificity pocket of matriptase, but not the S4 pocket as in the cases of hepatocyte growth factor activator·HAI-1 KD1 and matriptase·sunflower trypsin inhibitor-1 complexes. The long 60-loop of matriptase makes direct contact with HAI-1 but remains flexible even in the complexes, and its apex does not bind with KD1 tightly. The interactions between this unique 60-loop and KD1 may provide an opportunity to increase the specificity and inhibitory activity of KD1 for matriptase. Furthermore, comparison between KD1 and a homology model of HAI-1 KD2 rationalizes the structural basis of why KD1 but not KD2 is responsible for the inhibitory activity of HAI-1 against matriptase.

Structural Basis for Therapeutic Intervention of uPA/uPAR System

Urokinase-type plasminogen activator (uPA) is one of the two physiological serine proteases responsible for the activation of plasminongen to plasmin. uPA activity is regulated by its inhibitors (PAI-1 and PAI-2) and its receptor (uPAR), and an expanding list of their interacting proteins. In addition to plasminogen activation, this system also plays important roles in the regulation of many cellular processes including cell proliferation, adhesion and migration. It is beyond reasonable doubt that this enzyme system plays a central role in tumor biology and represents a high potential target for therapeutic intervention of tumor growth and metastasis. During the past fifteen years, crystal structures of uPA and its inhibitors have facilitated the development of uPA inhibitors. Many crystal structures of proteins in the uPA/uPAR system have also been reported recently, especially a series of structures of uPAR and its complexes with vitronectin and uPA, facilitating the development and evaluation of uPAR inhibitors. Recent progress on uPA inhibitors will be summarized in this article. The unique structural features and the druggable potentials of these new structures will also be discussed.

Quercetin-3-rutinoside Inhibits Protein Disulfide Isomerase by Binding to its b'x domain

Background: Quercetin-3-rutinoside is an inhibitor of protein disulfide isomerase, a potential target for antithrombotic therapy. Results: Quercetin-3-rutinoside induces a compact conformation in PDI and binds to PDI with an IC50 of about 10 μm. Conclusion: Quercetin-3-rutinoside interacts with the b′x domain of protein disulfide isomerase with a 1:1 stoichiometry. Significance: The b′x domain reverses the antithrombotic properties of quercetin-3-rutinoside in a thrombosis model in a live mouse. Quercetin-3-rutinoside inhibits thrombus formation in a mouse model by inhibiting extracellular protein disulfide isomerase (PDI), an enzyme required for platelet thrombus formation and fibrin generation. Prior studies have identified PDI as a potential target for novel antithrombotic agents. Using a fluorescence enhancement-based assay and isothermal calorimetry, we show that quercetin-3-rutinoside directly binds to the b′ domain of PDI with a 1:1 stoichiometry. The binding of quercetin-3-rutinoside to PDI induces a more compact conformation and restricts the conformational flexibility of PDI, as revealed by small angle x-ray scattering. The binding sites of quercetin-3-rutinoside to PDI were determined by studying its interaction with isolated fragments of PDI. Quercetin-3-rutinoside binds to the b′x domain of PDI. The infusion of the b′x fragment of PDI rescued thrombus formation that was inhibited by quercetin-3-rutinoside in a mouse thrombosis model. This b′x fragment does not possess reductase activity and, in the absence of quercetin-3-rutinoside, does not affect thrombus formation in vivo. The isolated b′ domain of PDI has potential as an antidote to reverse the antithrombotic effect of quercetin-3-rutinoside by binding and neutralizing quercetin-3-rutinoside.

The Binding Mechanism of a Peptidic Cyclic Serine Protease Inhibitor

Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding kinetics and thermodynamics by surface plasmon resonance and isothermal titration calorimetry. We found that upain-1 changes both main-chain conformation and side-chain orientations as it binds to the protease, in particular its Trp3 residue and the surrounding backbone. The properties of upain-1 are strongly influenced by the addition of three to four amino acids long N-terminal and C-terminal extensions to the core, disulfide-bond-constrained sequence: The C-terminal extension stabilises the solution structure compared to the core peptide alone, and the protease-bound structure of the peptide is stabilised by intrapeptide contacts between the N-terminal extension and the core peptide around Trp3. These results provide a uniquely detailed description of the binding of a peptidic protease inhibitor to its target and are of general importance in the development of peptidic inhibitors with high specificity and new inhibitory mechanisms.