Crystal Structure of the Parasite Protease Inhibitor Chagasin in Complex with a Host Target Cysteine Protease

The protozoan parasite Toxoplasma gondii relies on posttranslational modification, including proteolysis, of proteins required for recognition and invasion of host cells. We have characterized the T. gondii cysteine protease cathepsin L (TgCPL), one of five cathepsins found in the T. gondii genome. We show that TgCPL is the primary target of the compound morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS), which was previously shown to inhibit parasite invasion by blocking the release of invasion proteins from microneme secretory organelles. As shown by fluorescently labeled LHVS and TgCPL-specific antibodies, TgCPL is associated with a discrete vesicular structure in the apical region of extracellular parasites but is found in multiple puncta throughout the cytoplasm of intracellular replicating parasites. LHVS fails to label cells lacking TgCPL due to targeted disruption of the TgCPL gene in two different parasite strains. We present a structural model for the inhibition of TgCPL by LHVS based on a 2.0 Å resolution crystal structure of TgCPL in complex with its propeptide. We discuss possible roles for TgCPL as a protease involved in the degradation or limited proteolysis of parasite proteins involved in invasion.

mentioning

confidence: 81%

Toxoplasma gondii Cathepsin L Is the Primary Target of the Invasion-inhibitory Compound Morpholinurea-leucyl-homophenyl-vinyl Sulfone Phenyl

Larson

Parussini

Huynh

et al. 2009

“…The protein was expressed in a glutathione S-transferase fusion system (Amersham Biosciences) and purified by affinity and ion exchange chromatography as described earlier (16). The chagasin-containing fractions eluted in the final purification step were pooled, dialyzed against 100 mM Hepes buffer, pH 7.0, and concentrated to ϳ5 mg/ml using a Vivaspin column with a cut-off limit of 5 kDa (Vivascience, Lincoln, UK).…”

Section: Methodsmentioning

confidence: 99%

“…The properties of chagasin are similar to those of cystatins, but its structure is very different (15)(16)(17). Chagasin belongs to a new inhibitor family (MEROPS family I42) (1) and its mode of binding to the cysteine endopeptidases, cathepsin L and falcipain, has been recently elucidated (16,18,19). The present structures of the cathepsin B inhibitor complex provide a detailed atomic view of how the parasite protein inhibits a cysteine exopeptidase that may be in the first line of the host defense.…”

mentioning

confidence: 99%

Displacement of the Occluding Loop by the Parasite Protein, Chagasin, Results in Efficient Inhibition of Human Cathepsin B

Redzynia

Ljunggren

Abrahamson

et al. 2008

Self Cite

Cathepsin B is a papain-like cysteine protease showing both endo-and exopeptidase activity, the latter due to a unique occluding loop that restricts access to the active site cleft. To clarify the mode by which natural protein inhibitors manage to overcome this obstacle, we have analyzed the structure and function of cathepsin B in complexes with the Trypanosoma cruzi inhibitor, chagasin. Kinetic analysis revealed that substitution of His-110e, which anchors the loop in occluding position, results in 3-fold increased chagasin affinity (K i for H110A cathepsin B, 0.35 nM) due to an improved association rate (k on , 5 ؋ 10 5 M ؊1 s ؊1 ). The structure of chagasin in complex with cathepsin B was solved in two crystal forms (1.8 and 2.67 Å resolution), demonstrating that the occluding loop is displaced to allow chagasin binding with its three loops, L4, L2, and L6, spanning the entire active site cleft. The occluding loop is differently displaced in the two structures, indicating a large range of movement and adoption of conformations forced by the inhibitor. The area of contact is slightly larger than in chagasin complexes with the endopeptidase, cathepsin L. However, residues important for high affinity to both enzymes are mainly found in the outer loops L4 and L6 of chagasin. The chagasin-cathepsin B complex provides a structural framework for modeling and design of inhibitors for cruzipain, the parasite cysteine protease and a virulence factor in Chagas disease.

“…Like cystatins (10,11), the p41 fragment (14) and chagasin (15), clitocypins, and macrocypins bind to cysteine proteases along the whole active site cleft (Fig. 5).…”

Section: Discussionmentioning

confidence: 99%

“…The crystal structure of the p41 fragment bound to cathepsin L (14) has revealed similarity of the three binding loops to those of cystatins. The three-loop mode of binding is shared also by chagasin, the parasite inhibitor of papain-like proteases from Trypanosoma cruzi (15). Common to them all is the fact that the binding loops bind into the non-primed and primed substrate binding regions and occlude the catalytic cysteine residue but do not interact directly with it.…”

mentioning

confidence: 99%

Versatile Loops in Mycocypins Inhibit Three Protease Families

Renko

Sabotič

Mihelič

et al. 2010

Mycocypins, clitocypins and macrocypins, are cysteine protease inhibitors isolated from the mushrooms Clitocybe nebularis and Macrolepiota procera. Lack of sequence homology to other families of protease inhibitors suggested that mycocypins inhibit their target cysteine protease by a unique mechanism and that a novel fold may be found. The crystal structures of the complex of clitocypin with the papain-like cysteine protease cathepsin V and of macrocypin and clitocypin alone have revealed yet another motif of binding to papain like-cysteine proteases, which in a yet unrevealed way occludes the catalytic residue. The binding is associated with a peptide-bond flip of glycine that occurs before or concurrently with the inhibitor docking. Mycocypins possess a ␤-trefoil fold, the hallmark of Kunitz-type inhibitors. It is a tree-like structure with two loops in the root region, a stem comprising a six-stranded ␤-barrel, and two layers of loops (6 ؉ 3) in the crown region. The two loops that bind to cysteine cathepsins belong to the lower layer of the crown loops, whereas a single loop from the crown region can inhibit trypsin or asparaginyl endopeptidase, as demonstrated by site-directed mutagenesis. These loops present a versatile surface with the potential to bind to additional classes of proteases. When appropriately engineered, they could provide the basis for possible exploitation in crop protection.Inhibition of foreign protease activity is a widespread defense mechanism in plants against their pests, pathogens, and parasites (1). Protein inhibitors of proteases are present in a variety of plant tissues. They can be deployed alone or together with a variety of small molecules (2). It has been known for a long time that the expression of protease inhibitors is increased in injured plant leaves (3) and that their expression can be induced as a response to attack by insects or pathogens (2).Given the negative environmental effects of chemical pesticides used in crop protection, it is important to explore alternative approaches, such as the incorporation of genes encoding protease inhibitors into plants. Transgenic plants expressing various protease inhibitors have shown enhanced levels of insect resistance; however, the adaptive capacity of insect digestive proteases limits the use of single protease inhibitors (4, 5). The use of hybrid protease inhibitors with multiple inhibitory activity could, however, affect the functional properties of the fused inhibitors (6). Incorporation of genes encoding a range of protease inhibitors is to run the risk of deleterious modification of plants, but the use of a single protease inhibitor with versatile functionality could be the way forward.With these in mind, we have undertaken structural and mechanistic studies of the cysteine protease inhibitors clitocypin (Clt) 3 from mushroom Basidiomycetes Clitocybe nebularis and macrocypins (Mcp) from Macrolepiota procera. Based on the lack of sequence similarity to other protease inhibitors, they form separate protease inhibitor famil...