Article

Long-Range Communication between Different

Functional Sites in the Picornaviral 3C Protein
Highlights Authors
d Binding to a viral protein influences structural dynamics at Yan M. Chan, Ibrahim M. Moustafa,
another binding site Jamie J. Arnold, Craig E. Cameron,
David D. Boehr
d Ligand binding also influences the function of the other
binding site Correspondence
d Control of the energy landscape may regulate myriad ddb12@psu.edu
functions of this viral protein
In Brief
d Protein structural dynamics effectively increases the Binding of RNA and peptide to the
genomic information content picornaviral 3C protein leads to different
structural dynamic signatures,
influencing subsequent ligand and
protein complex interactions. Chan et al.
propose that these unique protein
fluctuations channel 3C toward distinct
functions important in the virus infection
cycle.

Chan et al., 2016, Structure 24, 509–517

April 5, 2016 ª2016 Elsevier Ltd All rights reserved
http://dx.doi.org/10.1016/j.str.2016.02.019
Structure
Article
Long-Range Communication between Different
Functional Sites in the Picornaviral 3C Protein
Yan M. Chan,1 Ibrahim M. Moustafa,2 Jamie J. Arnold,2 Craig E. Cameron,2 and David D. Boehr1,*
1Department of Chemistry, The Pennsylvania State University, University Park, PA 16802, USA
2Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA
*Correspondence: ddb12@psu.edu
http://dx.doi.org/10.1016/j.str.2016.02.019
SUMMARY
The 3C protein is a master regulator of the picornavi-
ral infection cycle, responsible for both cleaving viral
and host proteins, and interacting with genomic RNA
replication elements. Here we use nuclear magnetic
resonance spectroscopy and molecular dynamics
simulations to show that 3C is conformationally dy-
namic across multiple timescales. Binding of peptide
and RNA lead to structural dynamics changes at both
the protease active site and the RNA-binding site,
consistent with these sites being dynamically
coupled. Indeed, binding of RNA influences protease
activity, and likewise, interactions at the active site
affect RNA binding. We propose that RNA and pep-
tide binding re-shapes the conformational energy
landscape of 3C to regulate subsequent functions,
including formation of complexes with other viral
proteins. The observed channeling of the 3C energy
landscape may be important for regulation of the viral
infection cycle.
INTRODUCTION
Viral genomes tend to be quite compact but have evolved to
maximize their information content. One solution to the limited
coding capacities of RNA viruses is to combine multiple func-
tions into single proteins. The different functions of these viral
proteins may require them to interact with multiple viral RNA se-
quences and/or viral and host proteins (Bedard and Semler,
2004; Daijogo and Semler, 2011; Sola et al., 2011; Steil and Bar-
ton, 2009; Yin et al., 2007; Yu et al., 2013). In general, it is thought
that not all functions occur simultaneously and that ligand bind-
ing to one active/binding site may shut down the other. How this
communication occurs is not known.
The picornavirus 3C protein is a great model system for under-
standing the regulation of multiple functions in a viral protein. The
3C protein can also be found as a domain within the 3CD and
3BCD polyproteins (Cameron et al., 2010). The 3C(D) protein is
the major protease responsible for most of the polyprotein cleav-
age events (Cameron et al., 2010; Jore et al., 1988; Parsley et al.,
1999; Ypma-Wong et al., 1988) and is also important for regu-
lating the host cell’s response through cleavage of critical host
cell proteins (Chase et al., 2014; de Breyne et al., 2008;
Kuyumcu-Martinez et al., 2004; Rozovics et al., 2012; Walker
Structure 24, 509–517, April 5, 2016 ª2016 Elsevier Ltd All rights reserved 509
et al., 2013; Yalamanchili et al., 1997; Zhou et al., 2013). The
3C protein and/or its precursors also interact with cis-acting
replication elements (CREs) located within the viral genome (Steil
and Barton, 2009). Interactions with these RNA structures are
important for regulating replication and translation events (Blair
et al., 1998; Franco et al., 2005; Gamarnik and Andino, 1998;
Nayak et al., 2006; Parsley et al., 1997; Shih et al., 2004; Walker
et al., 1995; Yang et al., 2004). It is not completely clear if the two
functions (i.e., protease and RNA binding) of 3C are interrelated.
In Norovirus (NV) 3C-like protease, RNA binding was found to
impair the protease catalytic rate (Viswanathan et al., 2013),
but similar studies have not been done with picornaviral 3C pro-
teins. However, amino acid changes at the RNA binding site of
Enterovirus 71 (EV71) 3C decreased the protease activity, but
amino acid changes at the protease active site had little impact
on RNA binding (Shih et al., 2004). The protease activity of the
HRV 14 3C was likewise affected by amino acid changes at
the RNA-binding site (Walker et al., 1995).
The goals of the current research were to determine if protein/
peptide and RNA binding modulate the RNA-binding and prote-
ase activities of poliovirus (PV) 3C, respectively, and if so, to
delineate long-range interactions important for this regulation.
Regulation of the two functions of 3C through binding of the
other ligand may be important in the virus infection cycle. In
this article, we indeed demonstrate that RNA and peptide bind-
ing alter proteolytic activity and RNA binding, respectively. We
also identify millisecond conformational exchange events by nu-
clear magnetic resonance (NMR) R2 relaxation dispersion exper-
iments near the protease active site that are modulated by RNA
binding. These findings suggest that RNA and peptide binding
selects different subsets of 3C conformations, thereby regulating
subsequent 3C activities.
RESULTS
Mutual Regulation of the Two Functions of PV 3C
Previous studies indicated that RNA binding impairs the catalytic
rate of the NV 3C-like protease (Viswanathan et al., 2013). These
studies and others (Shih et al., 2004; Walker et al., 1995) sug-
gested that the two functions of 3C may be interrelated. To test
this hypothesis, we analyzed the protease activity and RNA-bind-
ing functions of PV 3C in the presence of the other ligand. In our
studies, we used two versions of 3C: proteolytically active
(3Cactive) and proteolytically inactive (3Cinactive) protein in which
the active site cysteine has been changed to an alanine,
and the remaining cysteine has been changed to a serine
(i.e., 3Cinactive has the substitutions C147A and C153S). Besides

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 cor-
responds 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 rep-
resented 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.
the protease activity, we do not expect significant functional and/
or structural dynamic differences between 3Cactive and 3Cinactive.
The C153S substitution is on the surface of the protein and not
near either the protease active site or the predicted RNA-binding
site. For RNA binding, we used either the oriI (origin of replication
Internal) RNA or the stem left (SL) region of the oriI RNA (Figure 1).
For the peptide, we used either the CA peptide, which corre-
sponds to the cleavage site between 2C and 3A of the polypro-
tein, or the AB peptide, which corresponds to the cleavage site
between 2A and 2B of the viral polyprotein (Figure 1).
To probe the effects of SL RNA binding on 3Cactive protease
activity, we adopted a fluorescence resonance energy transfer-
based protease assay in which proteolytic activity releases the
N-terminus attached fluorophore from the C terminus attached
quencher group (Hata et al., 2000) (Figure S1). We performed a
series of experiments with different SL RNA concentrations,
allowing us to perform ANOVA and post hoc Tukey honest signif-
icant difference (HSD) statistical tests. The presence of the
SL RNA had a small but statistically significant (i.e., ANOVA
p value
0.01; HSD p value for the largest SL RNA concentra-
tion
0.01) effect on the catalytic turnover rate constant (kcat)
when using a derivative of the CA peptide as substrate, but did
not have a statistically significant effect on the Michaelis-Menten
constant (KM) (Table 1).
We were also interested in probing the effect of peptide on
RNA binding. As such, we used fluorescence polarization exper-
iments to monitor RNA binding, which is labeled with fluorescein
510 Structure 24, 509–517, April 5, 2016 ª2016 Elsevier Ltd All rights reserved
Table 1. RNA Binding Has a Small Influence on 3C Protease
Activity
3Cactive:SL
RNA Ratio KM (mM) kcat (s1) kcat/KM (105 s1 M1)
1:0 62.73 ± 31.34 31.78 ± 8.76 5.07 ± 1.52
1:1 48.96 ± 20.71 34.04 ± 7.32 6.95 ± 1.73
1:2 53.61 ± 16.23 38.31 ± 6.08 7.17 ± 1.40
on the 30 end, to 3Cinactive in the absence and presence of pep-
tide. For these experiments, we monitored binding of SL RNA
and oriI RNA in the presence/absence of CA and AB peptides
(Figure 1). Again, we performed a series of experiments with
different concentrations of CA and AB peptides allowing us to
perform ANOVA and Tukey HSD statistical tests. The presence
of the CA peptide did not significantly alter the dissociation con-
stant (Kd) associated with SL RNA binding (Table 2; ANOVA
p value
0.61) but led to a small decrease in the oriI Kd (ANOVA
p value
0.02). In contrast, the presence of the AB peptide re-
sulted in more substantial changes in both SL and oriI RNA bind-
ing (Table 2). The presence of the AB peptide led to a decrease in
SL RNA-binding affinity (ANOVA p value
0.0001) but an in-
crease in oriI RNA-binding affinity (ANOVA p value
0.0006;
HSD p value for the largest peptide concentration
0.0005).
These results suggest that the effect of peptide binding on
RNA affinity is peptide-sequence dependent, and these effects
are also dependent on the sequence/structure of the RNA itself.
Poliovirus 3C Is a Monomer in Solution
Our studies indicated that RNA and peptide binding modulate
the protease and RNA-binding activities of PV 3C, respectively.
To gain more insight into the regulation of these two functions,
we characterized these interactions through NMR methodology.
The interpretation of our solution-state NMR experiments on PV
3C is dependent on the oligomeric state of the protein. Accord-
ing to dynamic light scattering (DLS) and NMR analyses, 3Cactive
and 3Cinactive are both monomeric in solution. The theoretical
translation diffusion coefficients for monomeric and dimeric 3C
are 9.9 3 1011 m2/s and 7.5 3 1011 m2/s, respectively (Amero
et al., 2008). The translational diffusion coefficient for 3Cinactive
was estimated to be 9.6 3 1011 m2/s by NMR and 9.3 3
1011 m2/s by DLS. Similarly, the translational diffusion coeffi-
cient for 3Cactive was estimated to be 9.9 3 1011 m2/s by
NMR and 9.4 3 1011 m2/s by DLS. Addition of the reducing
agent b-mercaptoethanol to 3Cactive (which has two Cys resi-
dues) did not substantially change the measured translational
diffusion coefficients (9.7 3 1011 m2/s by NMR and 9.3 3
1011 m2/s by DLS). These results are consistent with previous
NMR studies (Amero et al., 2008) and in contrast to the finding
that PV 3C crystallizes as a dimer (Mosimann et al., 1997). These
results are reminiscent of previous findings where NV 3C-like
protease was found to be a dimer in the X-ray crystal structure
(Zeitler et al., 2006) but a monomer in the NMR solution-state
structure (Takahashi et al., 2013).
RNA and Peptide Binding Lead to Long-Range NMR
Chemical Shift Perturbations
To gain some insight into the long-range effects of peptide and
RNA binding, we analyzed NMR chemical shift perturbations
Table 2. Peptide Binding Influences RNA-Binding Affinity (Amero et al., 2008; Claridge et al., 2009) and mutational studies
inactive (Andino et al., 1993; Blair et al., 1998; Hammerle et al., 1992;
3C :peptide Kd (Peptide)/Kd
RNA Peptide Ratio Kd for RNA (mM) (+Peptide) Leong et al., 1993; Nayak et al., 2006; Shih et al., 2004; Walker
et al., 1995) as being important for RNA binding. This cluster
SL CA 1:0 0.76 ± 0.14
included residues 81–89 on the h3 helix, residues on the N-ter-
1:1 1.23 ± 0.16 0.62
minal h1 helix (adjacent to h3), residues 29–32 on the b2 strand
1:5 0.87 ± 0.16 1.14 (nearby h3), and residues 156 and 157 on the b11 strand (nearby
AB 1:1 1.74 ± 0.44 0.44 h3). It should also be noted that upon binding SL RNA, some
1:5 1.63 ± 0.42 0.47 residues on the h3 helix showed two resonances (Figure 3;
oriI CA 1:0 12.83 ± 4.65 e.g., Asp85 and His89), which is consistent with conformational
1:2 10.84 ± 7.95 1.18 exchange on the slow NMR timescale. This effect is not due to
1:5 9.76 ± 3.50 1.31 the use of subsaturating concentrations of RNA; under these
conditions, we expect that 99.6% of 3C is bound with SL RNA
AB 1:1 9.01 ± 3.08 1.42
(based on Kd of 0.76 mM; see Table 2). The other cluster of res-
1:2 6.67 ± 0.86 1.92
idues show small chemical shift changes and correspond to res-
1:5 4.01 ± 0.43 3.20 idues nearby the protease active site, including Asn165 and
Phe170 on the C-terminal side and His40 and Glu63 on the
N-terminal side of the center cleft between the two b barrel sub-
(i.e., through 1H-15N heteronuclear single quantum coherence domains. These findings are similar to those for HRV-14 3C,
[HSQC] spectra) induced upon binding peptide or RNA. For which also suggested that structural changes around the prote-
these experiments, we only used the SL RNA because previous ase active site were induced upon binding RNA (Claridge et al.,
DLS experiments had indicated that, in the presence of the full 2009).
29-nt oriI RNA, PV 3C forms a multimer, which would severely For CA peptide binding, residues associated with substantial
complicate NMR analysis; this does not occur with the SL RNA chemical shift changes also appear in two clusters (Figure 2B).
(Amero et al., 2008). One cluster is located in the region between the two b barrel sub-
We identified resonances with appreciable chemical shift domains where the catalytic triad lies, including residues 23–25,
changes as those showing chemical shift changes at least one 40–41 on the N-terminal b barrel subdomain, and residues 102,
SD above the average chemical shift change across all 116, 132, 137, 142, 148, 165, 168–171 on the C-terminal b barrel
resonances. Appreciable chemical shift changes induced by subdomain. The other cluster is nearby the predicted RNA-bind-
SL RNA binding are associated with amino acid residues ing site, including Arg176 and Thr180 on the C-terminal h4 helix,
in two clusters (Figure 2A). One cluster is close by the KFRDI and His89 on the h3 helix adjacent to the C-terminal h4 helix.
motif, which was identified by previous NMR experiments Binding of AB peptide resulted in a similar pattern of chemical

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
Ddcombined = (DdH2 + (DdN/5)2)1/2 where DdH
and DdN are the chemical shift differences be-
tween 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 mM 3Cinactive in
the presence/absence of 215 mM SL RNA or
215 mM CA peptide using a buffer consisting of
10 mM HEPES (pH 7.5) and 50 mM NaCl.
See also Figure S2.

Structure 24, 509–517, April 5, 2016 ª2016 Elsevier Ltd All rights reserved 511

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 mM 3Cinactive
in the presence/absence of 215 mM SL RNA or 215 mM CA peptide using a
buffer consisting of 10 mM HEPES (pH 7.5) and 50 mM NaCl.
See also Figure S3.
shift perturbations as that induced by the CA peptide, although
the chemical shift perturbations were generally of lower magni-
tude (Figure S2).
512 Structure 24, 509–517, April 5, 2016 ª2016 Elsevier Ltd All rights reserved
RNA and Peptide Select Different Sets of 3C
Conformations
It is intriguing that both SL RNA and peptide binding induced
chemical shift changes to resonances belonging to two clusters
of amino acid residues (Figure 2C). One cluster is nearby the pre-
dicted ligand-binding site, and the other cluster is nearby the
predicted binding site of the other ligand. These findings are
intriguing considering that we have shown that RNA/peptide
binding can affect the interactions of 3C with the other ligand
(i.e., peptide/RNA; Tables 1 and 2). To gain more insight into
these potential cooperative effects, we formed the binary com-
plexes with either RNA or peptide and then titrated in peptide
or RNA, respectively. Remarkably, some of the resonances
associated with these ternary complexes were at different chem-
ical shift positions than that observed for either of the binary
complexes (including catalytic residues His40 and Glu71) (Fig-
ure 3A). In fact, many of these residues were associated with
two resonances, again suggestive of conformational exchange
on the slow NMR timescale. Perhaps even more remarkable
was the finding that the chemical shift positions of these reso-
nances depended on whether SL RNA or CA peptide was
added first (Figure 3B). Many of the residues associated with
multiple resonances in the ternary complexes (Figure 3C) are
near the 3C dimer interface as observed by crystallographic
studies (Mosimann et al., 1997) and/or near interfaces proposed
to be important for interacting with other PV proteins (Shen et al.,
2008).
RNA Binding Selects a Different Ensemble of
Conformations
The appearance of two resonances for some amino acid resi-
dues in the RNA binary or ternary complexes suggested that
there are multiple conformations of 3Cinactive in solution
exchanging on the slow NMR timescale. To probe this further,
we performed NMR R2 relaxation dispersion experiments, which
report on protein structural dynamics on the micro- to milli-
second timescale (Loria et al., 2008) (Figure 4A). Unfortunately,
we were not able to perform these experiments in the presence
of CA peptide because we could not achieve saturating condi-
tions with the peptide due to its low solubility.
One parameter that can be gleaned from the R2 relaxation
dispersion experiments is Rex, which is the contribution to R2
from conformational exchange and indicates those regions of
the protein undergoing conformational exchange on the micro-
to millisecond timescale. For 3C without ligand, the resonances
with substantial Rex (i.e., >5 s1) are associated with residues at
the center cleft near the protease active site and residues near
the N-terminal h1 helix (Figures 4B and S3). The corresponding
amino acid residues include Tyr6, Asn14, and Ile15 on the N-ter-
minal h1 helix, Phe25 on the b2 strand adjacent to the N-terminal
h1 helix, Asn105 on the loop adjacent to the h1 helix, Lys175,
Phe179, and Gln181 on the h4 helix of the C terminus, Leu37
and residues 60–75 on the N-terminal b barrel subdomain, and
Gln122, Gly123, Ala133, Met160, Ala171, and Ala172 on the
C-terminal b barrel subdomain. Similar patterns of conforma-
tional exchange were observed when 3Cinactive was bound with
SL RNA, although there were a few additional regions that
showed conformational exchange (including residues 13, 100–
103, 108, 116, 150, and 162).

Data from the R2 relaxation dispersion experiments can also
give more quantitative information about the kinetics and ther-
modynamics of conformational exchange. Assuming conforma-
tional exchange between two conformations (say conformations
A and B), R2 relaxation dispersion experiments can yield the ex-
change rate constant kex that is the sum of the forward (kA/B) and
reverse (kB/A) rate constants, the populations of the exchange
conformations (pA, pB), and the dynamic chemical shift difference
between the exchanging conformations (Du = duA  duB) (Loria
et al., 1999). Generally, experimental data for different residues
are fit with global kex and pA/pB values, and residue-specific
Du values. The global kex and pB values for 3Cinactive without
RNA (kex = 480 ± 22 s1, pb = 0.020 ± 0.001) and with SL RNA
(kex = 518 ± 19 s1, pB = 0.023 ± 0.001) were very similar. It should
be noted that residues undergoing conformational exchange are
generally not those that show substantial chemical shift pertur-
bations upon binding SL RNA (Figure 2A; Table S1), so compar-
isons of Du values can give some insight into how the binding of
SL RNA affects the nature of the higher energy protein conforma-
tion (Figure 4D). Many of the Du values correlate between the apo
and RNA-bound states, however, there are a group of residues in
which the Du values for the SL RNA binary complex are substan-
tially higher than the Du values for 3Cinactive without RNA. These
residues tend to be spatially close to the protease active site (Fig-
ure 4C; residues shown in blue). Many of these residues, or res-
idues nearby, show two or more resonances in the RNA/peptide
ternary complexes (Figure 3). It is also interesting that SL RNA
binding increased kcat by a similar amount (
1.2-fold) as it
increased the back rate constant for the R2 relaxation dispersion
experiments (kB/A = 9.6 s1 and 11.9 s1 for without and with SL
RNA, respectively). Altogether, these results suggest that binding
of SL RNA selects for a different subset of 3C conformations, and
these conformations may interact with peptides at the active site
differently than 3C without RNA.
Nanosecond Dynamics of 3C Are Also Modulated by RNA
Binding
The NMR experiments indicated that 3C is highly dynamic on the
micro- to millisecond timescale. We also probed 3Cactive struc-
Structure 24, 509–517, April 5, 2016 ª2016 Elsevier Ltd All rights reserved 513
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 s1 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 (Du) 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 Du > 4.5 ppm are
not plotted. Residues with substantially different
Du 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.
tural dynamics on the nanosecond timescale using all-atoms
molecular dynamic (MD) simulations and experimentally through
further NMR experiments. For the MD simulations, the 3Cactive
monomer extracted from the crystal structure (PDB: 1L1N) was
immersed in a box filled with water solvent and subjected to
100 ns MD simulation. Analysis of the MD trajectory revealed
that, on average, the 3Cactive structure did not show substantial
deviations from the starting crystal structure during simulation
(root-mean-square deviation [RMSD] = 1.52 Å). Nevertheless,
the amino terminal residues 1–13 showed large deviations (Fig-
ure 5A); the first five residues appeared more extended toward
solution in comparison with the crystal structure. Many of these
residues were identified as being associated with substantial
chemical shift changes upon binding RNA.
To obtain information on the structural dynamics of individual
amino acids, we performed per residue RMSD analysis (per-
RMSD) across the last 50 ns of the trajectory (Figure 5B). In
this analysis, the average positional deviations of individual res-
idues, including heavy side-chain atoms, were calculated rela-
tive to the starting structure. The N-terminal residues 1–13
revealed the largest deviations with per-RMSD values in the
range 2.7–14.1 Å. Most of the 3C residues, 75% of the total res-
idues, exhibited per-RMSD values lower than 1.6 Å. The residues
that showed relatively large deviations are distributed across the
entire 3C structure with peaks at positions 22, 31, 45, 52, 65, 81,
93, 113, 130, 143, 167, and 180 (Figure 5B). Dynamics of resi-
dues around positions 22, 65, 130, 143, and 167 are expected
to affect the binding of the peptide substrate, whereas dynamics
of residues around positions 31, 52, 81, and 113 could affect
RNA binding. Mapping of the per-RMSD values on the 3C struc-
ture revealed that almost all residues involved in peptide binding
that showed large chemical shift perturbations exhibited low-
to-moderate amplitude nanosecond dynamics (Figure 5C),
whereas, residues involved in RNA binding exhibited moder-
ate-to-large amplitude nanosecond dynamics (Figure 5C).
To help experimentally verify some of the MD results, we also
attempted to collect T1 longitudinal and T2 transverse relaxation
times, but unfortunately, the experimental data were not of suffi-
cient quality for Lipari-Szabo model-free analysis (Lipari and

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.
Szabo, 1982a, 1982b). Nonetheless, we were able to gain some
qualitative insight by analyzing the steady-state 1H-15N hetero-
nuclear Overhauser effects (hetNOEs) (Figure S4), which tend
to correlate with order parameters (S2). According to these re-
sults, the most flexible region is the loop between the b6 strand
and the a3 helix that connects the two b barrel subdomains. The
MD simulations also show that this region is dynamic on the
nanosecond timescale (Figure 5).
With the NMR experiments, we were also able to evaluate
dynamic changes induced by the presence of RNA. Addition of
SL RNA led to some local changes to the hetNOE values for
3Cinactive. Phe25 and Gly29 on the interface of the N-terminal b
barrel subdomain and Val162 and Ala172 on the interface of
the C-terminal b barrel subdomain had increased hetNOE values
in the presence of SL RNA, suggestive of decreased pico-
to nanosecond dynamics, whereas Gln65, Glu71, Glu121,
Arg134, Leu136, and Ala173 had decreased hetNOE values
(i.e., increased dynamics) in the presence of SL RNA.
DISCUSSION
Viral genomes are typically small. One way viruses maximize the
information content in their genomes is to encode multifunctional
proteins like the 3C protease. One way to rationalize the multiple
functions of 3C is to envision an ensemble of conformations,
whose members are responsible for different functions (Boehr,
2012). For example, different conformations may be responsible
for interacting with different RNA or protein binding partners;
similar scenarios have been invoked for ubiquitin (Lange et al.,
2008) and other proteins (Boehr et al., 2009) to help explain
how these proteins interact with such divergent binding partners.
514 Structure 24, 509–517, April 5, 2016 ª2016 Elsevier Ltd All rights reserved
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 resi-
dues (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 indi-
cated 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-moder-
ate dynamics during simulation. In this view, the
peptide-binding site is at the front. (Right) A
Here, we have shown that 3C fluctuates into different conforma-
tions across multiple timescales, and binding partner interac-
tions alter the thermally accessible conformations to regulate
3C function.
NMR chemical shift perturbation analyses indicated that RNA/
peptide binding leads to structural dynamics changes at both the
predicted binding site and around the binding site of the other
ligand for 3Cinactive (Figure 2). Some residues were associated
with multiple resonances when RNA/peptide was bound, sug-
gesting conformational exchange on the slow NMR timescale
(Figure 3). Intriguingly, many of these residues had different
chemical shift positions depending on the order of RNA/peptide
binding (Figure 3). RNA binding also induced changes in the
pico- to nanosecond (Figure S4) and micro- to millisecond time-
scale dynamics of 3Cinactive (Figure 4). With RNA bound, residues
around the protease active site fluctuated into a different confor-
mation, as assessed by the R2 relaxation dispersion studies (Fig-
ure 4). The higher energy conformation might be important for
interacting with protein substrates, according to a conforma-
tional selection mechanism (Boehr et al., 2009; Kumar et al.,
2000; Miller and Dill, 1997; Tsai et al., 1999). These results sug-
gest that binding of RNA (peptide) selects a different subset of
conformations, which then affects how peptide (RNA) will subse-
quently interact with 3C (Figure 6). In fact, peptide binding influ-
ences RNA affinity (Table 2) and RNA affects proteolytic activity
(Table 1).
Binding of peptide/RNA thus appears to change the structural
dynamics of 3C, which can then regulate how 3C interacts with
other binding partners. In this context, it was especially inter-
esting that the AB peptide had a more substantial effect on
RNA binding than the CA peptide (Table 2). These differences

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.
may be related to the different functions of the P2 and P3
proteins within the polyprotein (Figure S1). The AB peptide is
representative of the cleavage sites within the P2 domain of
the polyprotein. The P2 proteins are important for host-protein
interactions (2A) and generation of vesicles and replication
organelles (2B and 2BC) (Gradi et al., 1998; Rust et al., 2001;
Trahey et al., 2012; Ventoso et al., 1998). The CA peptide is
representative of the cleavage site that releases P3 proteins
from P2 proteins; P3 proteins are most notable for their roles in
genome replication and post-translational processing (Cameron
et al., 2010). These findings suggest that many of the P2 and P3
protein functions are separated temporally, and so their proteo-
lytic release by 3C(D) may be regulated differentially by RNA
interactions.
Changes to which conformations are thermally accessible
may also affect interactions with other viral or host proteins.
Many of the residues that show differences in the pico- to nano-
second (Figure S4) and micro- to millisecond (Figures 4 and S3)
dynamics upon binding RNA have been implicated as being
important for peptide binding and protease activity (Blair et al.,
1996; Mosimann et al., 1997). Other residues are important in
forming higher order complexes with other viral proteins. For
example, a structural model involving a 3C dimer and a 3D
monomer binding to oriI RNA has been proposed to be important
for initiation of RNA synthesis (Shen et al., 2008). Many of the 3C
residues that form intersubunit interactions within this complex
are conformationally dynamic on the millisecond timescale, as
shown either by the R2 relaxation dispersion experiments (Fig-
ure 4) or by the presence of multiple resonances in the ternary
complexes (Figure 3), including residues important for 3C-3D in-
teractions such as Phe25, Lys108, and Asn165 and residues
important for 3C-3C interactions such as Lys60, Ala61, and
Asn69. These results suggest that conformational fluctuations
on the millisecond timescale may be preparing 3C to form a
dimer, as it does in the X-ray crystal structure, or interact with
3D and potentially other binding partners.
Structure 24, 509–517, April 5, 2016 ª2016 Elsevier Ltd All rights reserved 515
It should also be kept in mind that the 3C protein can be found
as part of the 3BCD and 3CD polyproteins. The 3BC(D) protein
may be involved in initiation of genome replication; 3CD is
thought to be the major protease and the major CRE-binding
protein (Cameron et al., 2010). In fact, 3CD generally has higher
proteolytic activity and different protease specificity compared
with 3C (Parsley et al., 1999; Ypma-Wong et al., 1988). However,
the reasons for these differences are unclear. The crystal struc-
ture of 3CD appears simply to be a composite of the 3C and 3D
domains with no additional protein interactions that may help to
explain these functional differences (Marcotte et al., 2007). The
covalent attachment of the 3D domain to the C terminus of 3C
may alter the internal motions of 3C to influence RNA-binding
ability and protease activity. The ability to fit the R2 relaxation
dispersion data to global kinetic and thermodynamic parameters
suggests that the millisecond motions in 3C are concerted, and
so changes in the N and C termini would likely lead to dynamic
changes elsewhere in the protein, especially at the active site
and RNA-binding site. It has been previously proposed that dy-
namic fluctuations may be responsible for functional differences
between 3C and 3CD (Cameron et al., 2009). Dynamic regions in
3C may be involved in other biomolecular interactions; targeted
studies of these dynamic regions may reveal novel biomolecular
interactions important in regulating the multifunctional roles of
3C in viral infection.
EXPERIMENTAL PROCEDURES
Protein Overexpression and Purification
Following transformation of Escherichia coli BL21(DE3) pCG1 cells with
plasmids encoding 3Cactive or 3Cinactive (i.e., C147A/C153S), cells were
overexpressed using autoinducible minimal media as described previously
(Studier, 2005). Protein purification was carried out as previously described
(Amero et al., 2008). Protein concentration was measured by a NanoDrop
spectrophotometer (Thermo Scientific) or SpectroMax M2 spectrophotometer
(Molecular Devices) using the theoretical extinction coefficient at 280 nm of
8,940 M1 cm1. More detailed information about protein overexpression
and purification can be found in the Supplemental Experimental Procedures.
Dynamic Light Scattering Experiments
DLS experiments were performed with a Viscotek 802 Instrument (Malvern
Instruments). Data were analyzed using the mass model on OmniSIZE 3.0
(Malvern Instruments).
NMR Analyses
1
H-15N HSQC NMR spectra were recorded at 25 C on either a 600 MHz or
850 MHz Bruker Avance III spectrometer equipped with a 5 mm inverse detec-
tion, triple resonance (1H/13C/15N), single-axis gradient TCI cryoprobe. 15N
R2 relaxation dispersion datasets were obtained simultaneously on both 600
MHz and 850 MHz Bruker Avance III spectrometers equipped with TCI cryo-
probes. 15N R2 relaxation dispersion experiments were performed using the
pulse sequences as described previously (Loria et al., 1999). Parameter fittings
were performed using the GLOVE program (Sugase et al., 2013). More detailed
information can be found in the Supplemental Experimental Procedures.
Pulsed-field gradient experiments were performed with the 850 MHz Bruker
instrument equipped with a diffusion probe using pulse sequences described
previously (Price, 1997). NMR titration and diffusion experiments were per-
formed in 10 mM HEPES (pH 7.5), 50 mM NaCl at 298 K. R2 relaxation disper-
sion experiments were performed in 25 mM potassium phosphate (pH 8.0) and
150 mM NaCl at 293 K.
Fluorescence-Based Assays
Fluorescence polarization experiments were performed at 25 C on a Beacon
2000 fluorescence polarization analyzer (Life Technologies). Up to 20 mM of
3Cinactive was incubated at 25 C for 30 s with 0.2 nM 30 -fluorescein-labeled SL
or oriI RNA. The buffer used was 10 mM HEPES (pH 7.5), 10 mM NaCl. The
fluorescence protease assay for 3Cactive was previously described (Hata
et al., 2000). Assays were performed at 30 C on a SpectraMax M2 microplate
reader using a 96-well microtiter plate. The buffer used for the assays con-
sisted of 10 mM HEPES (pH 7.5), 50 mM NaCl. The volume of the assays
was 200 ml. Peptide was dissolved in DMSO immediately before use; the
DMSO concentration made up no more than 5% (v/v) of the final sample
mixture. Peptide concentration was determined by weight and volume. Data
were obtained with excitation at 328 nm and emission at 393 nm. More details
can be found in the Supplemental Experimental Procedures.
MD Simulations
MD simulations were performed similar to those described previously (Mous-
tafa et al., 2011) using the AMBER12 package (Case et al., 2005; Roe and
Cheatham, 2013) with an amber 99SB force field (Hornak et al., 2006). The 3C
monomer (chain A) of the crystal structure PDB: 1L1N was used as the initial co-
ordinates for the simulation. The 3C molecule was immersed in a truncated oc-
tahedron solvent box filled with TIP3P water molecules; the distance between
any protein atom and the edge of the solvent box was kept at 20 Å. The charge
of the simulated structure was neutralized by adding the appropriate number of
counter ions. The all-atom NPT simulation was carried out at 300 K, and the
Berndsen thermostat was used to control the temperature during simulation.
The total simulation time was 100 ns using a 1 fs integration time step; any
bonds involving H atoms were constrained using the Shake algorithm. The
non-bonded interactions were calculated under periodic boundary conditions
using a cutoff distance of 9 Å; the electrostatic interactions were calculated us-
ing particle mesh Ewald method. Analysis of the MD trajectory was done as
described previously (Moustafa et al., 2011), utilizing the last 50 ns of the trajec-
tory using Ptraj and Cpptraj of the AMBER package (Roe and Cheatham, 2013).
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
four figures, and one table and can be found with this article online at http://
dx.doi.org/10.1016/j.str.2016.02.019.
AUTHOR CONTRIBUTIONS
Conceptualization, Y.M.C., I.M.M., C.E.C., and D.D.B.; Methodology, Y.M.C.,
I.M.M., J.J.A., C.E.C., and D.D.B.; Software, Y.M.C. and I.M.M.; Formal Anal-
ysis, Y.M.C., I.M.M., and D.D.B.; Investigation, Y.M.C. and I.M.M.; Data Cura-
tion, I.M.M., C.E.C., and D.D.B.; Writing – Original Draft, Y.M.C., I.M.M., and
D.D.B.; Writing – Review & Editing, Y.M.C., I.M.M., J.J.A., C.E.C., and
D.D.B.; Visualization, Y.M.C., I.M.M., and D.D.B.; Supervision, J.J.A., C.E.C.,
and D.D.B.; Project Administration, C.E.C. and D.D.B.; Funding Acquisition,
C.E.C. and D.D.B.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Adriano Z. Zambom for advice with the sta-
tistical analyses and Drs. Julia Fecko and Neela Yennawar for help with prelim-
inary biophysical experiments. The authors would also like to thank Drs. Nihal
Altan-Bonnet, Ann Cali, and Peter Takvorian for discussions on viral proteins.
This study was supported by grants from the US NIH (R01AI091985 to Nihal
Altan-Bonnet, Ann Cali, Peter Takvorian, and D.D.B.; R01AI053531 to
C.E.C.; R01AI104878 to D.D.B.).
Received: October 11, 2015
Revised: February 17, 2016
Accepted: February 26, 2016
Published: April 5, 2016
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