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. 2007 Apr;81(7):3583-96.
doi: 10.1128/JVI.02306-06. Epub 2007 Jan 24.

Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-dependent RNA Polymerase

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Free PMC article

Crystal Structure of Poliovirus 3CD Protein: Virally Encoded Protease and Precursor to the RNA-dependent RNA Polymerase

Laura L Marcotte et al. J Virol. .
Free PMC article

Abstract

Poliovirus 3CD is a multifunctional protein that serves as a precursor to the protease 3C(pro) and the viral polymerase 3D(pol) and also plays a role in the control of viral replication. Although 3CD is a fully functional protease, it lacks polymerase activity. We have solved the crystal structures of 3CD at a 3.4-A resolution and the G64S fidelity mutant of 3D(pol) at a 3.0-A resolution. In the 3CD structure, the 3C and 3D domains are joined by a poorly ordered polypeptide linker, possibly to facilitate its cleavage, in an arrangement that precludes intramolecular proteolysis. The polymerase active site is intact in both the 3CD and the 3D(pol) G64S structures, despite the disruption of a network proposed to position key residues in the active site. Therefore, changes in molecular flexibility may be responsible for the differences in fidelity and polymerase activities. Extensive packing contacts between symmetry-related 3CD molecules and the approach of the 3C domain's N terminus to the VPg binding site suggest how 3D(pol) makes biologically relevant interactions with the 3C, 3CD, and 3BCD proteins that control the uridylylation of VPg during the initiation of viral replication. Indeed, mutations designed to disrupt these interfaces have pronounced effects on the uridylylation reaction in vitro.

Figures

FIG. 1.
FIG. 1.
The poliovirus 3CD structure. (a) There are two molecules of 3CD in the asymmetric unit. In this stereo view, one molecule is red and one is blue. The 3C and 3D domains are tethered together by a polypeptide linker region (upper black arrow). Within each molecule of 3CD, there is no direct contact between the 3C and 3D domains, except through the linker. The length of the polypeptide linker and the position of the protease site within the 3C domain (lower purple arrow) preclude intramolecular cleavage of 3CD. Regions of greatest variability between 3CD and the 3Cpro and 3Dpol structures are shown in green. (b) Representative electron density from an omit-phased 2FoFc map contoured at 1.4σ is shown in stereo. This view shows density surrounding a proposed Zn2+ site at an interface between the 3D domains of two symmetry-related copies of 3CD, in gray and light blue. The Zn2+ ion appears to be tetrahedrally coordinated by Asp446, His453, His455, and Cys464. Density for the uppermost 3D domain comes from an α helix, while the density in the lower 3D domain includes β strands.
FIG. 2.
FIG. 2.
Hydrogen-bonding interactions around the N terminus of 3Dpol G64S, shown in stereo. An omit-phased 2FoFc map contoured at 1.4σ shows density including the G64S mutation. Ser64 forms a hydrogen bond with Glu2, which in turn interacts with Gln4 of the N terminus. Gly1 maintains contacts with Ala239. Additional hydrogen bonding interactions are formed between the backbone of the N terminus and the backbone of a neighboring β strand.
FIG. 3.
FIG. 3.
Structure of the polypeptide linkers between the 3C and 3D domains of the two molecules of 3CD in the asymmetric unit. An omit map contoured at 1.4σ shows weak density in the linker regions, though the overall path of the backbone is clear. The linker regions of the two 3CD molecules in the asymmetric unit differ in structure, confirming that the linker is only weakly ordered. The two linkers are shown in similar orientations, with the 3C domain on the left and the 3D domain on the right.
FIG. 4.
FIG. 4.
Key interfaces formed by 3CD-3CD interactions. (a) The three labeled interfaces are formed between symmetry-related molecules of 3CD. Interfaces D-C and D-C′ are each formed between the 3C domain of one molecule and the 3D domain of another. Interface D-D is formed between two 3D domains that are related by twofold crystallographic symmetry. Green, yellow, and blue ribbons come from three different 3CD molecules. The thumb, palm, and fingers region of the green molecule are labeled. The N terminus of the yellow 3C domain is indicated. (b) Interface D-C is shown in stereo. Key residues and hydrogen bonds are indicated. (c) A portion of interface D-D is shown in stereo, with some key residues and hydrogen bonds indicated. Two symmetry-related copies of the putative Zn2+ coordination site are present, with Zn2+ ions indicated as gray spheres. Pro88 from interface D-C is included in both panel b and panel c as an aid to orientation.
FIG. 5.
FIG. 5.
Comparison of the 3CD and 3Dpol structures at the N terminus, the active site, and the fingers domain shown in stereo. (a) The structure of the active site of the polymerase remains mostly unchanged between 3CD (light gray) and 3Dpol (dark gray). Notable residues of the 3Dpol active site are colored: Asp238, Ala239, Leu241, and Asn297 are magenta, the GDD motif (Asp328) is cyan, the N-terminal fingers are green, and the middle finger is orange. (b) One of the largest positional differences between 3CD and 3Dpol occurs within the fingers region of the 3D domain (3CD residues 226 to 251; 3Dpol residues 43 to 68). 3CD is dark gray with Gly184 in yellow; 3Dpol is light gray and green with Gly1 also in green and motif A in magenta. Note that the N-terminal G184 of 3CD is removed from the binding cleft, whereas Gly1 of 3Dpol is buried. Only slight differences are seen in motif A (residues 238 to 241 of 3Dpol), magenta in 3Dpol.
FIG. 6.
FIG. 6.
Hydrogen-bonded linkage between amino-terminal residues (blue), active site residues (red and yellow), and the fingers domain (gray) in several viral RNA-dependent RNA polymerases (RdRps). The RdRps of poliovirus, FMDV and HRV14, form hydrogen-bonding networks, connecting the fingers domain with the N terminus and active site residues. These interactions are absent in PV 3CD. Structural motif A is red; structural motif C is yellow (18).
FIG. 7.
FIG. 7.
3BCD and its cleavage products: substrates and stimulatory factors for uridylylation. Large ovals represent the viral protein domains; wavy lines represent polypeptide chains. The cre RNA is predicted to have a stem-loop structure and is shown in gray. The purple and yellow rectangles represent the D-D and D-C interfaces, respectively, as they are seen in the 3CD crystal structure (shown in Fig. 5). The binding of additional copies of the 3C and/or 3D domains and the involvement of additional interfaces cannot be ruled out.
FIG. 8.
FIG. 8.
Proposed model of the uridylylation complex. (a) Several poliovirus 3Dpol residues appear to stabilize VPg (red) in its putative binding pocket. Lys172 and Arg179 (green) are critical for stabilizing Tyr3 of VPg in the active site. Residues in blue and red correspond to residues of FMDV that position VPg in its binding pocket. (b) The D-D and D-C interfaces may be responsible for stabilizing interactions between 3BC(D) (yellow and red) and a 3D polymerase molecule (green). VPg (red) has been modeled into the 3D domain using the FMDV-VPg structure as a template (12). These binding interactions might be relevant to the stimulation of VPg uridylylation by either 3C or 3CD or to the binding of uncleaved 3BC or 3BCD as substrates for uridylylation.
FIG. 9.
FIG. 9.
How the structure of the uridylylation complex might affect biological function. Large ovals represent the viral protein domains; wavy lines represent polypeptide chains. The cre RNA is predicted to have a stem-loop structure and is shown in gray. The purple and yellow rectangles represent the D-D and D-C interfaces, respectively, as they are seen in the 3CD crystal structure. Aggregation of 3CD may compete with the formation of the uridylylation complex (top panel). 3CD might dissociate either intact or after it has been cleaved (middle panel). Interfaces D-D and D-C can coexist with interface I (red rectangle, bottom panel, left); however, the formation of interface D-C′ (white rectangle) would be in steric conflict with interface I (bottom panel, right). Interface II (not shown) would conflict with interface D-C.

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