1. Volume 24, Issue 4, Pages 509-517 (April 2016)
Long-Range Communication between Different Functional Sites in the Picornaviral 3C Protein 
Yan M. Chan, Ibrahim M. Moustafa, Jamie J. Arnold, Craig E. Cameron, David D. Boehr 
Structure 
Volume 24, Issue 4, Pages (April 2016)
DOI: /j.str
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2. Figure 1 The Poliovirus 3C Protease Has Multiple Functions
(A) The structure of the 3C protease (PDB: 1L1N) showing the locations of the proposed RNA binding site (in green spheres) and the catalytic triad (in black spheres).
(B) 3C interacts with viral RNA control sequences including the internal origin of replication (oriI) site. The stem left (SL) RNA that is used in these studies corresponds to the nucleotide sequence shown in blue.
(C) 3C catalyzes the hydrolysis of peptide bonds in the viral polyprotein, including between 2C and 3A, and 2A and 2B. These cleavage sites are represented by the CA and AB peptides, respectively. The two C-terminal Arg residues (underlined) are added to the peptide to increase solubility, but they are not predicted to interact with 3C.
See also Figure S1.
Structure  , DOI: ( /j.str )
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3. Figure 2
RNA and Peptide Binding Perturb the Chemical Environment around the Other Ligand's Binding Site
(A and B) 1H-15N HSQC spectra were compared with 3Cinactive without and 3Cinactive bound with (A) SL RNA or (B) CA peptide. NMR chemical shift perturbations were calculated using the equation Δδcombined = (ΔδH2 + (ΔδN/5)2)1/2 where ΔδH and ΔδN are the chemical shift differences between 3C with and without the appropriate ligand for the backbone amide proton and nitrogen, respectively.
(C) Residues showing substantial chemical shift perturbations in the presence of RNA and/or peptide are plotted as spheres on the 3C X-ray crystal structure (PDB: 1L1N), where those with chemical shift perturbations one and two SDs above the average are shown respectively in light blue (orange) and darker blue (red) for SL RNA (peptide) binding. Residues that show substantial chemical shift pertubations for both SL RNA and peptide binding are shown in purple. NMR spectra were collected at 298 K with ∼215 μM 3Cinactive in the presence/absence of 215 μM SL RNA or 215 μM CA peptide using a buffer consisting of 10 mM HEPES (pH 7.5) and 50 mM NaCl.
See also Figure S2.
Structure  , DOI: ( /j.str )
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4. Figure 3
The Conformational Ensemble of 3C Is Different when Bound with Both Peptide and RNA Compared with when It Is Bound with Only One Ligand
(A) Spectral overlays of specific resonances from 1H-15N HSQC spectra collected for ligand-free 3Cinactive (black), and 3Cinactive bound with SL RNA (blue) or CA peptide (red) or both (purple).
(B) The NMR spectra of the ternary complex bound with peptide and SL RNA also depends on the order of ligand binding. The resonances are colored purple and cyan for when SL RNA and CA peptide are added first, respectively.
(C) Locations of residues associated with two or more resonances in the ternary complexes bound with peptide and RNA are shown as magenta colored spheres. NMR spectra were collected at 298 K with ∼215 μM 3Cinactive in the presence/absence of 215 μM SL RNA or 215 μM CA peptide using a buffer consisting of 10 mM HEPES (pH 7.5) and 50 mM NaCl.
See also Figure S3.
Structure  , DOI: ( /j.str )
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5. Figure 4
The Binding of RNA Selects for a Different Subset of 3C Conformations
(A–C) Example NMR R2 relaxation dispersion curves collected at 1H Larmor frequencies of 600 (black) and 850 (red) MHz (A). Error bars indicate estimated uncertainties in R2. Residues displaying Rex values greater than 5 s−1 at 850 MHz for (B) ligand-free 3Cinactive and (C) 3Cinactive bound with SL RNA are plotted as colored spheres.
(D) Comparison of the dynamic chemical shift changes (Δω) derived from the R2 relaxation dispersion curves between ligand-free 3Cinactive and SL RNA-bound 3Cinactive. It should be noted that some residues that show Δω > 4.5 ppm are not plotted. Residues with substantially different Δω in the presence/absence of SL RNA are also plotted as blue spheres in (C). The R2 relaxation dispersion experiments were conducted at 295 K using a buffer consisting of 25 mM potassium phosphate (pH 8.0) and 150 mM NaCl.
See also Table S1.
Structure  , DOI: ( /j.str )
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6. Figure 5
Nanosecond Dynamics Nearby the Protease Active Site and RNA-Binding Site
(A) Shown is the average 3Cactive structure (gray), calculated from the last 50 ns of the MD simulation, superimposed on the 3C crystal structure (light blue, PDB: 1L1N). Apart from the N-terminal residues (aa 1–13), the two structures superimpose well with an RMSD of 1.52 Å.
(B) The calculated per-RMSD across the last 50 ns of the trajectory is plotted as a function of residue. Residues corresponding to the peaks are indicated by red arrows and labeled. The N-terminal residues appeared to show the largest amplitude dynamics during the simulation.
(C) The per-RMSD in (B) is mapped onto the average 3C structure. Residues corresponding to the highest 25% of per-RMSD data (>1.6) are colored red, residues corresponding to the lowest 25% of per-RMSD data (<0.8) are colored green, and residues with per-RMSD values in the range 0.8–1.6 are colored gray. (left) Residues that showed largest perturbations in chemical shifts due to peptide binding are displayed as spheres and labeled; these residues, with the exception of His168, revealed a small-to-moderate dynamics during simulation. In this view, the peptide-binding site is at the front. (Right) A different view in which the RNA-binding site is at the front. Residues that showed largest perturbations in chemical shifts upon RNA binding are displayed as spheres and labeled; these residues revealed moderate-to-large dynamics during the simulation.
See also Figure S4.
Structure  , DOI: ( /j.str )
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7. Figure 6 Conformational Channeling in the 3C Protein
Schematics of the conformational energy landscapes on 3C in the absence and presence of peptide and RNA ligands. Importantly, the ligand-binding order determines the lowest energy conformation in the ternary complex, as also suggested by the chemical shift perturbations in Figure 3. This channeling of the free energy landscape may also affect how 3C interacts with other viral and host factors.
Structure  , DOI: ( /j.str )
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