Similar to the previously reported structures of the 3C proteases, the HRV-C15 3C protease contains a chymotrypsin-like fold that can be divided into two major domains and one soft linker (Table 1) (Mosimann et al. 1997; Matthews et al. 1999; Anand et al. 2002; Yang et al. 2003; Lee et al. 2009; Lu et al. 2011). In domain Ⅰ, seven β-strands (βaI–βgI) surround the αB short helix and make up the core of the domain (Fig. 1). The domain Ⅱ is mainly composed of nine β-strands (βaII–βiII), which is further stabilized by the neighboring N-terminal helix αA (Fig. 1). The two main domains are linked by αC and a long loop which are positioned on the opposite side of the catalytic center (Fig. 1). Three highly conserved residues, H40, E71 and C147, located in the center of the substrate-binding groove formed by the two major domains, play key roles in the catalysis (Fig. 1). The long soft linker situated on the opposite side enables the substrate-binding groove to be more flexible in terms of size and orientation (Fig. 1).
Name 3C 3C in complex with rupintrivir Data collection Resolution (Å) 50–2.15 (2.23–2.15) 50.00–2.05 (2.12–2.05) Unique reflections 13, 578 (1332) 16, 114 (1496) Space group C2221 C2221 Cell dimensions a (Å) 52.1 52.9 b (Å) 94.7 95.6 c (Å) 98.1 98.9 α (°) 90 90 β (°) 90 90 γ (°) 90 90 Redundancy 7.5 (6.4) 3.1 (3.0) Completeness (%) 99.8 (99.5) 92.1 (90.2) aRmerge 0.069 (0.328) 0.060 (0.263) I/σ(I) 35.6 (8.1) 22.8 (7.0) Refinement Resolution(Å) 2.15 2.05 No. reflections 13, 546 14, 812 bRwork/cRfree 0.210/0.253 0.184/0.223 No. of non-H atoms Protein 1498 1, 419 Mean B-factor (Å2) 31.5 28.0 Ramachandran statistics (%) Most favored 97.2 98.3 Allowed 2.8 1.7 Outliers 0.0 0.0 R.m.s.deviations Bond lengths (Å) 0.010 0.008 Bond angles (°) 1.089 1.086 Values in parentheses are for highest-resolution shell.
aRmerge ∑hkl∑i|I(hkl)i- < I(hkl)>|/∑hkl∑iI(hkl)i,
cRfree was calculated for a test set of reflections (5%) omitted from the refinement.
Table 1. Data collection and refinement statistics.
Figure 1. Overall structure of the HRV-C15 3C protease. Cartoon representation of the structure of HRV-C15 3C protease is shown with domain Ⅰ, domain Ⅱ and the linker colored green, red and blue, respectively. The α-helices are marked αA to αD and the β-strands are labeled βaI to βgI in domain Ⅰ and βaII to βiII in domain Ⅱ according to their occurrence along with the primary structure. The catalytic triad (H40, E71, and C147) is shown as orange sticks and labeled.
Rupintrivir is a specific 3C inhibitor which was designed based on the protein structure (Matthews et al. 1999). As reported previously, this irreversible inhibitor could be divided into five groups—a lactam (P1), a fluorophenylalanine (P2), a Val (P3), a 5-methyl-3-isoxazole (P4), and an α, β-unsaturated ester (P1′) (Matthews et al. 1999; Lu et al. 2011). Rupintrivir fills itself into the catalytic pocket of 3C protease and further stabilizes the binding with a covalent linkage to C147, one of the three key catalytic residues (Fig. 2A and Table 1). Four of the five groups of the inhibitor: P1, P2, P3 and P1′, and the covalent bond can be traced based on clear and unambiguous density, while the P4 group of rupintrivir is totally invisible in the complex, suggesting that a simplified inhibitor lacking the P4 group could still possibly block the cleavage efficiently (Fig. 2A).
Figure 2. Structure of the HRV-C15 3C-rupintrivir complex. A Overall structure of the 3C-rupintrivir complex. 3C protease is colored and labeled as Fig. 1. The rupintrivir is shown as sticks. The covalent bond formed between the inhibitor and the protease is highlighted with an arrow. The inset corresponds to zoomed-in view of boxed region and illustrates the features of the inhibitor. B Line representation of the superimposition of the 3C protease in free and inhibitorbound forms colored in cyan and blue, respectively. C Cartoon representation of the superimposition of the 3C proteases of HRV-2, HRV-C15, EV71 and CVA16, colored orange, blue, red and green, respectively. Insets correspond to zoomed-in views of boxed regions and illustrate the structural differences with secondary elements labeled.
The overall structures of 3C protease of HRV-C15 in free and inhibitor-bound forms are very similar (Fig. 2B). Superimposition of the two structures of 3C protease yields an RMSD of 0.239 Å for the overlapping Cα atoms and reveals that rupintrivir blocks the catalytic activity of 3C without changing the overall conformation of the protease. Interestingly, although the overall structures of complexes of 3C protease and rupintrivir of HRV-2, HRV-C15, EV71 and CVA16 are very similar, subtle deviations in the structures have been observed during the superposition (Fig. 2C). Two antiparallel β-strands (βaI and βbI) of domain Ⅰ move "down" towards domain Ⅱ by about 4 Å when compared with the same two β-strands of the other three complexes (Fig. 2C). The βaII-βbII loop of domain Ⅱ (the loop connecting the βaII and βbII strands) varies in conformation and the βaII-βbII loops of HRV-2 and HRVC15 tend to bend towards the catalytic center, which is different from those observed for 3C proteases from EV71 and CVA16 (Fig. 2C). These conformational changes of HRV-C15 3C protease lead to a relatively more closed S1' subsite (the binding site of rupintrivir P1′ group) and reduce the volume of this substrate-binding pocket.
3C proteases of HRV-2, HRV-C15, EV71 and CVA16 share a chymotrypsin-like fold in the overall structure and a highly conserved catalytic center. Rupintrivir binds to the different proteases in a similar manner. Besides the obvious covalent linkage, some distinct characteristics have been observed from the conformation of the inhibitor while comparing these complexes. In different complexes, P1 and P3 groups of the inhibitors are in the same position while the P1′ and P2 groups rotate to adapt to the different microenvironments of the binding subsites (Fig. 3). Rupintrivir shows a new conformation upon binding to HRV-C15 3C protease when compared to its binding to 3C proteases of HRV-2, EV71 and CVA16. The P1′ group rotates ~ 54° relative to its position in the complex with HRV-2 and stretches out into the solvent, which is similar to that observed in the complexes of the inhibitor with EV71 and CVA16 (Fig. 3). The P2 group sticks into the S2 subsite of HRV-C15 in a manner similar to that observed for HRV-2 3C-rupintrivir complex and is tilted 21° from the P2 groups of rupintrivir binding with 3C proteases of EV71 and CVA16 (Fig. 3). An "intermediate" conformation of rupintrivir appears in the complex of HRV-C15 and displays some characteristics of both conformers. The new conformation of rupintrivir and the differences in various binding modes might help explain the differences in the inhibitor's binding affinities and EC50 values for different viruses.
Figure 3. Rupintrivir adopts a new conformation. The inhibitor (shown in sticks) and the 3C proteases (shown in cartoon) of HRV-2, HRVC15, EV71 and CVA16 are colored orange, blue, red and green, respectively. The transparency of the cartoon representation of 3C proteases of HRV-2, EV71 and CVA16 is 0.7. Insets correspond to zoomed-in views of boxed regions and illustrate the conformational changes of the inhibitor with the rotation angles labeled.
In the complex of HRV-C15 3C protease with rupintrivir, microenvironment in the pocket somewhat impacts the inhibitor's conformation, especially while binding to S1' and S2 subsites. In the S1' subsite of HRV-C15 3Crupintrivir complex, the 4 Å -movement of βaI and βbI reduces the volume of the subsite remarkably when compared with those of HRV-2, EV71 and CVA16 (the structure of CVA16 3C protease is almost identical to that of EV71 and therefore omitted in the following structural analysis) (Figs. 2 and 4A). Residues with long side chains in the β-strands, such as K22 and F25, compress the S1' subsite further and leave insufficient space for holding the P1′ group of rupintrivir. Steric clashes would have occurred if the P1′ group inserted itself into the subsite in HRV-C15 3C-rupintrivir complex just like that observed in the complex of HRV-2. Residue N22 of HRV-2 3C protease and Q22 of EV71 3C protease stretch their side chains out to the solvent and bring much less stress than K22 of HRVC15 3C protease does (Fig. 4A). The highly conserved residue F25 follows the movement of the two β-strands to encroach on the subsite with its long side chain (Fig. 4A). Interestingly, although the S1 pocket of HRV-C15 3C protease is smaller than that of EV71, the rotation angle of the inhibitor's P1′ group in its complex with HRV-C15 3C is smaller than that observed for EV71 3C-rupintrivir complex (Figs. 3 and 4A).
Figure 4. Structural comparison of the S1' and S2 subsites of 3C proteases of HRV-2, HRV-C15 and EV71. In A and B, rupintrivir is shown as sticks and the 3C proteases are shown in surface representations with the key residues and secondary structural elements shown as sticks and cartoon, respectively. The polar interaction between R39 and P2 group of the inhibitor in the complex of EV71 is colored yellow in B.
Two states of the S2 subsite, fully open and half-closed, have been reported for the 3C protease of HRV-2 and EV71, respectively. Accordingly, the P2 group of rupintrivir has been observed to insert itself into S2 subsite when open, and reach out of the subsite when half-closed (Matthews et al. 1999; Lu et al. 2011). Interestingly, the P2 group of rupintrivir still inserts itself into the pocket while the S2 subsite of HRV-C15 3C protease stays half-closed, which indicates that the binding mode of P2 group of rupintrivir is not determined by the state of the subsite (Figs. 3 and 4B). N130 is conserved in HRV-2 and HRVC15, and the orientations of its side chain control the state of the S2 subsite together with K69/D69 located on the other side. The state of the subsite switches from fully open to half-closed as the side chain of N130 swaps from "downside" (like that observed for N130 of HRV-2 3C protease) to "upside" (as observed for K130 of EV71 3C protease) (Fig. 4B). The polar interaction between R39 and rupintrivir P2 group makes the P2 group turn ~ 21° and extend into the solvent since the long side chain of R39 stretches out of the subsite (Figs. 3 and 4B). The conserved T39 residues in rhinoviruses do not interact with rupintrivir so that the P2 group could remain inserted inside the subsite (Figs. 3 and 4B).
The size-reduced S1' and the half-closed S2 subsites are not the unique features of the enterovirus A 3C proteases (such as EV71 and CVA16). The S1' subsite of HRV-C protease is even smaller than that of EV71 as well as CVA16 and the half-closed S2 subsite is also observed in the protease of HRV-C. Unexpectedly, the states of the S2 subsite have no impact on the conformation of the P2 group of rupintrivir. Although most 3C proteases share a highly conserved overall structure, the differences in the residues could result in variations in the local microenvironments of the subsites. Consequently, the conformation of the inhibitor changes to adapt to the variations in the subsites.