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

Abstract

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.

The Occluding Loop of Cathepsin B Prevents Its Effective Inhibition by Human Kininogens

“…61,62 This is in contrast to papain and cathepsins L and S, which bind family 1 and 2 cystatins according to a one-step mechanism. [63][64][65][66] Likewise, the present experimental kinetics values, corresponding to the tight-binding inhibition of His110Ala cathepsin B by both kininogens, strongly suggest that the disruption of the salt bridge between His110 and Asp22 results in increased mobility of the occluding loop and a subsequent improved accessibility to the active site as previously discussed in detail by Pavlova et al 36 and Redzynia et al, 35 leading to a single, one-step reaction mechanism similar to that observed for occluding loop-lacking cathepsins interacting with stefins and cystatins. Human stefin B, similarly to kininogens and their isolated inhibitory domains, binds tightly to papain and cathepsin L and more weakly to cathepsin B. Stefin B also has a lower affinity to cathepsin B than ovocystatin or stefin A.…”

“…35 ClusPro 81 was used to dock D2 onto cathepsin B. ClusPro applies a series of filters to identify complexes with good surface complementarity and low desolvation energies and selects docked complexes with the broadest energy wells. Briefly, the top 20,000 complexes with the best shape complementarity are trimmed down to 2000 complexes based on electrostatic and desolvation free energies.…”

“…34 Since the occluding loop in cathepsin B is flexible, it is inhibited by cystatins (low micromolar). 35 While general features of the binding and inhibition of cathepsin B by stefins (family 1) and cystatins (family 2) have been well established, 36 fewer studies have been reported for kininogens (family 3 cystatins). LMWK cystatin-like domains (D2 and D3 domains) bind each cathepsin molecule independently and D3 has a larger k on , resulting in greater inhibitor potential.…”

β-Trefoil inhibitors – from the work of Kunitz onward

“…In addition to the mycocypins, the mode of inhibitor binding to the C1 protease has been described for the cystatin (I25) (Stubbs et al , 1990 ) and thyropin (I31) families (Gunčar et al, 1999 ) as well as for chagasin (I42) (Wang et al , 2007 ;Redzynia et al , 2008 ). The inhibitors from these families have completely different folds but still use different loops to occlude the active-site residues.…”

“…The inhibitors from these families have completely different folds but still use different loops to occlude the active-site residues. In contrast to two loops in clitocypins, proteins such as cystatins, thyropins, and chagasin provide three loops, which fi ll the active-site cleft (Stubbs et al , 1990 ;Gunč ar et al, 1999 ;Wang et al , 2007 ;Redzynia et al , 2008 ).…”