Figure 1.
Sequence alignment of AcCYS to the other cystatins.
The sequence of AcCYS is compared with taro, rice, potato, human cystatin F, chicken egg white cystatin, human stefin A, and human stefin B. The characteristic motifs for putative interaction with target cysteine protease are marked by green asterisks. The unique motif, LARFAVxExN of phytocystatins is denoted by the red dots on top of the sequence. The α-helix and β-sheet regions are colored in red and blue, respectively.
Table 1.
Structural statistics of 20 lowest-energy structures of AcCYS.
Figure 2.
(A) The ensemble of 20 best structures of the inhibitory domain of AcCYS (res 41 to 135) is superimposed. The α-helical, β-sheet, and loop region is color coded in red, green, and grey respectively. (B) Ribbon representation of the inhibitory domain of AcCYS structure shows a αβ roll structure made up of one α-helix and four anti-parallel β-strands, β1–β4. Three regions which contain the highly conserved motifs are labeled as CM1–3. Side chains of these highly conserved residues including, G45, Q89VVSG, and W120 are shown in spheres. These diagrams were generated using the structural visualization program PYMOL.
Figure 3.
Hydrophobic clusters of AcCYS on the interface of α-helix and β-sheet.
Hydrophobic side chains that participate in stabilizing the compact structure of AcCYS are labeled and shown in sticks (A) and in spheres (B).
Figure 4.
Equilibrium unfolding of AcCYS and AcCYS_DL.
The GdnHCl-induced unfolding curves of AcCYS (▴) and AcCYS_DL (○) were monitored by CD at the wavelength of 222 nm. The transition curve was quantified and the corresponding value of for AcCYS and AcCYS_DL is 4.4, and 4.5 Kcal/mol, respectively.
Figure 5.
The Lineweaver-Burk plots for the inhibition of papain by AcCYS and AcCYS-DL.
The inhibition of papain by AcCYS and AcCYS-DL is shown in (A) and (B) respectively. The initial rates of cleavage of a fluorogenic substrate, Z-Phe-Arg-7-amido-4-methylcoumarin (FRAMC) hydrochloride by papain were obtained spectrofluorometrically with excitation and emission wavelengths at 346 and 450 nm, respectively. Line 1 represents papain activity (2 nM) in the absence of inhibitor. Line 2–4 show the enzyme activity in the presence of 40, 60, 80 pM cystatin, respectively. Both AcCYS and AcCYS-DL strongly inhibited papain with inhibitory constant KI of 2.0±0.2×10−10 M and 1.4±0.1×10−10 M, respectively.
Figure 6.
Complex formation of AcCYS_DL and papain.
(A) 15N-1H HSQC spectra of AcCYS-DL alone (red) and in association with papain (blue). The residues with most significant chemical shift perturbation are labeled. (B) The overall chemical shift perturbation, Δδresidue of AcCYS_DL upon association with papain was plotted against residue number. Δδresidue of each residue was calculated as described in Materials and Methods. The average of Δδresidue for all residues of AcCYS_DL upon association with papain was calculated and shown as the yellow horizontal solid line (0.066 ppm). The pink and green horizontal solid lines represented two and three fold averaged Δδresidue, respectively.
Figure 7.
Local secondary structural rearrangements of AcCYS when associated with papain.
(A) CSI plot of free AcCYS_DL. (B) CSI plot of AcCYS_DL upon association with papain. (C) H/D exchange of amide protons of V90, V91, E60, D61, and L62 in the bound form (▴) and in the free state of AcCYS DL (○).
Figure 8.
Structural model of AcCYS/papain complex.
(A) The complex structure of AcCYS/papain was simulated by ZDOCK. AcCYS is shown in yellow ribbons. The binding site of AcCYS is shown in yellow spheres. The surface of papain is shown in light grey. The active site of papain including C25, H159, and N175 residues are shown in red spheres. (B) Complex structure of AcCYS/papain determined by restrained molecular dynamics simulations. AcCYS is shown in light blue ribbons. The G44–G45 and Q89–V91 of AcCYS shown in spheres completely block the active site of papain. The first turn of α-helix, residue 56–58 collapses. (C) AcCYS/papain complex stabilized by intermolecular forces. The backbone of papain is shown in yellow line. An intermolecular H-bond between G45 amide proton of AcCYS and the side chain carboxyl of D158 of papain and an intermolecular salt bridge between the carboxyl of D48 of AcCYS and K156 of papain are formed in the simulated complex structure (shown in sticks). (D) A new hydrophobic cluster between AcCYS and papain. The side chain of Y47 of AcCYS (shown in light blue spheres) as well as Y61, Y66, and W69 residues of papain (shown in yellow spheres) form an intermolecular aromatic cluster.