Nonnative interactions in coupled folding and binding processes of intrinsically disordered proteins.

Abstract

Proteins function by interacting with other molecules, where both native and nonnative interactions play important roles. Native interactions contribute to the stability and specificity of a complex, whereas nonnative interactions mainly perturb the binding kinetics. For intrinsically disordered proteins (IDPs), which do not adopt rigid structures when being free in solution, the role of nonnative interactions may be more prominent in binding processes due to their high flexibilities. In this work, we investigated the effect of nonnative hydrophobic interactions on the coupled folding and binding processes of IDPs and its interplay with chain flexibility by conducting molecular dynamics simulations. Our results showed that the free-energy profiles became rugged, and intermediate states occurred when nonnative hydrophobic interactions were introduced. The binding rate was initially accelerated and subsequently dramatically decreased as the strength of the nonnative hydrophobic interactions increased. Both thermodynamic and kinetic analysis showed that disordered systems were more readily affected by nonnative interactions than ordered systems. Furthermore, it was demonstrated that the kinetic advantage of IDPs ("fly-casting" mechanism) was enhanced by nonnative hydrophobic interactions. The relationship between chain flexibility and protein aggregation is also discussed.

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Nonnative Interactions in Coupled Folding and Binding
Processes of Intrinsically Disordered Proteins
Yongqi Huang1,2,3, Zhirong Liu1,2,3*
1State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing, China, 2Center
for Theoretical Biology, Peking University, Beijing, China, 3Beijing National Laboratory for Molecular Sciences, Peking University, Beijing, China
Abstract
Proteins function by interacting with other molecules, where both native and nonnative interactions play important roles.
Native interactions contribute to the stability and specificity of a complex, whereas nonnative interactions mainly perturb
the binding kinetics. For intrinsically disordered proteins (IDPs), which do not adopt rigid structures when being free in
solution, the role of nonnative interactions may be more prominent in binding processes due to their high flexibilities. In
this work, we investigated the effect of nonnative hydrophobic interactions on the coupled folding and binding processes
of IDPs and its interplay with chain flexibility by conducting molecular dynamics simulations. Our results showed that the
free-energy profiles became rugged, and intermediate states occurred when nonnative hydrophobic interactions were
introduced. The binding rate was initially accelerated and subsequently dramatically decreased as the strength of the
nonnative hydrophobic interactions increased. Both thermodynamic and kinetic analysis showed that disordered systems
were more readily affected by nonnative interactions than ordered systems. Furthermore, it was demonstrated that the
kinetic advantage of IDPs (‘‘fly-casting’’ mechanism) was enhanced by nonnative hydrophobic interactions. The relationship
between chain flexibility and protein aggregation is also discussed.
Citation: Huang Y, Liu Z (2010) Nonnative Interactions in Coupled Folding and Binding Processes of Intrinsically Disordered Proteins. PLoS ONE 5(11): e15375.
doi:10.1371/journal.pone.0015375
Editor: Vladimir N. Uversky, Indiana University School of Medicine, United States of America
Received August 12, 2010; Accepted August 18, 2010; Published November 4, 2010
Copyright: ? 2010 Huang, Liu. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Ministry of Science and Technology of China (No. 2009CB918500) (http://www.most.gov.cn/) and the National Natural
Science Foundation of China (No. 20973016 and No. 10721403) (http://www.nsfc.gov.cn/Portal0/default124.htm). The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: LiuZhiRong@pku.edu.cn
Introduction
Deciphering how physical interactions affect protein behavior is
fundamental to structural and functional biology. As a first
approximation, interactions presented in the native state (native
interactions) dominate in processes such as protein folding and
binding, resulting in a funnel-like energy landscape with minimal
frustration [1–3]. Under such conditions, the Go ¯-model [4,5] has
been widely adopted to generate valuable insights into protein
folding and binding [6–10]. In realistic systems, however, the
existence of nonnative interactions is inevitable. The effects of
nonnative interactions on protein folding have been demonstrated
in many experimental studies [11–23]. Nonnative interactions
perturb the denatured state ensemble and thus affect the
equilibrium stability [11,12], lead to the accumulation of the on-
pathway or off-pathway intermediate states [13–15], and most
importantly, moderate the protein folding kinetics by perturbing
the transition state [16,17,20,21]. Nonnative interactions also act
as a major driving force in the rapid collapse of an unfolded
protein during the early stages of the folding process, which is
important in preventing proteins from aggregating [22]. In the
phosphorylation-activation process of a signal protein (nitrogen
regulatory protein C), the disruption of some native contacts was
compensated by the transient formation of nonnative interactions
[23]. For protein binding processes, nonnative interactions have
been recognized to be important in the initial formation of the
non-specific encounter complexes, where long-range electrostatic
interactions increase the diffusion process by the ‘‘steering effect’’,
and then short-range hydrophobic interactions facilitate the
formation of the final specific complexes by a two-dimensional
search on the surface [24–27].
The effects of nonnative interactions on protein folding have
been extensively studied by simulations and analytical theories
[28–45]. An all-atom simulation suggested that ,20–25% of the
energy in the transition state arose from nonnative contacts [28].
Nonnative hydrogen bonds in a simulation on the helix-coil
transition were found to be most populated around the transition
temperature [29]. Mutation can also change the population of
nonnative contacts [30]. In general, the existence of nonnative
interactions may influence protein stability and folding kinetics
[31]. A lattice model simulation showed that nonnative interac-
tions have little effect on protein stability, but would accelerate
protein folding and thus give rise to the W-values that are negative
or larger than unity [32]. Further detailed analytical and
simulation studies found that the folding rate generally enhances
initially as the nonnative interactions increase, but drops rapidly
when the nonnative interactions are larger than a critical value
[33–36]. The importance of nonnative interactions on folding
kinetics was also validated by a direct comparison between
simulation and experiment for the SH3 protein [37]. In the novel
designed protein Top7 [46], it was revealed that the noncooper-
ative folding kinetics is caused by both native topology and
nonnative interactions [38,39]. For proteins with more compli-
cated topologies, e.g., knotted proteins, it was suggested that
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nonnative interactions play an essential role in the correct
formation of the knots [40]. Despite the perturbation on folding
kinetics, the protein folding mechanism is usually robust with
respect to nonnative interactions [43,44]. Compared with the
extensive studies on protein folding, theoretical investigations on
protein binding are relatively rare [47–51]. The mechanisms of
the electrostatic rate enhancement via lowering the transition state
energy and the dimensionality-reducing effect by non-specific
binding to DNA and cell membranes are well understood;
however, the effects of short-range hydrophobic interactions on
general protein–protein binding processes remain unresolved.
The influences of nonnative interactions may be more
prominent in intrinsically disordered proteins (IDPs). IDPs are a
special family of proteins that lack unique tertiary structures under
physiological conditions, either along the entire chain or in
particular regions [52–55]. IDPs were predicted to be enriched in
both prokaryotic and eukaryotic genomes [56,57] and perform
various functions, including transcription and translation regula-
tion, cellular signal transduction, protein modifications, and
molecular assemblies. In particular, IDPs have been shown to be
associated with human diseases such as cancer, cardiovascular
disease, amyloidosis and neurodegenerative diseases [58]. Al-
though disordered when alone in solution, in many cases, IDPs
undergo conformational transitions to folded states upon binding
their biological targets to perform functions [59]. Go ¯-like models
have been successfully applied to study the coupled folding and
binding processes of IDPs [9,49,60–63]. This approach has
contributed important insights into the characteristics of IDPs,
e.g., the kinetic advantages in molecular recognition for IDPs
through the ‘‘fly-casting’’ mechanism [64]. Considering the
significant chain flexibility of IDPs, IDPs are expected to possess
more nonnative interactions in the folding and binding processes
than conventional ordered proteins. It would be important to
investigate the different effects of nonnative interactions on IDPs
and ordered proteins and whether taking into account the
nonnative interactions would change the principles of IDPs
elucidated by native-centric models.
In this paper, we conducted computer simulations to study how
nonnative hydrophobic interactions affect the binding thermody-
namics and kinetics of IDPs, and how these effects are related to
the chain flexibility which distinguishes IDPs from the ordered
proteins.
Results
IDPs are readily trapped into non-specific states
To investigate the effects of nonnative hydrophobic interactions
on the coupled folding and binding of IDPs, we modified a coarse-
grained Go ¯-like model of IDPs [62] to include a sequence-
dependent hydrophobic-polar (HP) component which accounts for
the nonnative hydrophobic interactions [37] (see Materials and
Methods). We used our model to simulate the binding of the
phosphorylated kinase-inducible domain (pKID) of the transcrip-
tion factor cAMP response-element binding protein to the kinase
inducible domain interacting domain (KIX) of the cAMP
response-element binding protein. The pKID domain is a well
characterized IDPs which folds upon binding to KIX [26,65,66].
In the model, a parameter a was introduced to scale the strength of
the intra-molecular native interactions and thus tune the chain
flexibility of pKID [62], while another parameter KHPwas used to
describe the strength of the nonnative hydrophobic interactions.
The influences of nonnative hydrophobic interactions on the
binding free energy of the pKID-KIX complex and their interplay
with the chain flexibility are summarized in Figure 1. Here, we
used the fraction of native contacts between the two proteins (Qb)
as a reaction coordinate to depict the binding process. The
nonnative hydrophobic interactions were found to mainly stabilize
the partially bound states with moderate Qbvalues, but had little
influence on either the unbound or bound states. Over-strong
nonnative hydrophobic interactions can even trap an intermediate
state centered at Qb,0.2 (Figures 1A,B). Interestingly, our results
clearly showed that the effect of nonnative hydrophobic
interactions on a disordered system (with a small SQf(free)T) was
more remarkable than that on an ordered system (with a large
SQf(free)T). To quantitatively measure the influence on the
equilibrium properties, we divided the conformation space into
three states: the unbound state (U), intermediate state (I), and
bound state (B) (Figure 1B). The population of the non-specific
intermediate state increased as the strength of the nonnative
hydrophobic interactions KHPwas increased, and the disordered
system exhibited a more remarkable increase (Figure 2A).
Although the nonnative hydrophobic interactions frustrated the
binding free-energy landscapes, its influence on the stability of the
complex was rather small because the free-energy difference
between the bound and unbound states showed negligible change
with KHP(Figure 2B). When analyzing the transition temperature
(Tm, defined as the temperature corresponding to the peak in the
heat capacity curve [62]), the same conclusion was reached: Tm
remained constant with increasing KHP(Figure 2C).
Flexibility promotes dynamic and extensive non-specific
interactions
To further characterize the nonnative hydrophobic interactions
along the binding process, we examined the number of nonnative
contacts (NHP) of pKID in the free state, binding intermediate
state, and the bound state. In the free state, the radius of gyration
(Rg) showed that pKID underwent a minor compression in the
presence of nonnative hydrophobic interactions (Figure 3A).
Compared with the total number of intramolecular native contacts
in the bound state (i.e., 25), the number of nonnative contacts was
rather small (Figure 3B). For the most disordered system,
SNHP(free)T was only ,1.0 even when KHP=1.5, and as expected,
the ordered system showed no nonnative contacts. The small
number of nonnative contacts observed for pKID in the free state
explains the insensitivity of the equilibrium binding free-energy
with respect to the strength of nonnative hydrophobic interactions
(Figure 2B) because, by definition, the native bound state was
affected only by native interactions. This observation was also
consistent with results on protein folding which showed that the
number of nonnative contacts of globular proteins is usually
significantly smaller than the number of native contacts in the
folded state [29,39]. Therefore, intramolecular native contacts
were not affected in the free state (Figure 3C). In contrast, in the
binding process the intermediate state showed a considerable
number of nonnative contacts, particularly for the disordered
system (Figures 4A–C). Under the same strength of the nonnative
hydrophobic interactions, the nonnative contact number of the
intermediate state showed a remarkable decrease with decreasing
chain flexibility (Figure 4D). Compared with the intermediate
state, the bound state possessed fewer nonnative hydrophobic
contacts (Figure 4E). This feature strongly indicates that chain
flexibility promotes dynamic and extensive non-specific interac-
tions during the binding process and will have a significant effect
on the binding kinetics. When correlating the nonnative contact
number of the intermediate state to the strength of the nonnative
interactions, a sharp increase in the nonnative contact number
appeared (Figure 4F), which leads to misbinding states (Figure 2A).
Nonnative Interactions in IDPs
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Binding rate is accelerated by nonnative hydrophobic
interactions
It was established from the Go ¯-models that IDPs possess faster
binding rates than ordered proteins via the ‘‘fly-casting’’
mechanism whichwouldfacilitate
[60,62,64]. Considering the findings in protein folding studies
that nonnative interactions can accelerate or decelerate the folding
rates [32–37,39] and the above observation that nonnative
contacts are more prevalent in IDPs than in ordered proteins, it
would be intriguing to explore whether the kinetic advantage of
IDPs will be smeared by the nonnative interactions. We simulated
the binding kinetics of the pKID-KIX complex under various
strengths of nonnative interactions, and the results are summarized
in Figure 5. In a similar manner to protein folding, nonnative
interactions have a nonlinear effect on the binding kinetics, i.e., the
binding rate initially increased and then slowed sharply as the
strength of the nonnative hydrophobic interactions increased, and
this observation was independent of chain flexibility (Figures 5A,B).
The existence of the critical strength of nonnative interactions was
also observed in the work of Turjanski et al. [60]. In the binding-
rate increase region, the disordered system possessed a greater
binding rate than the ordered system, showing a kinetic advantage
in the binding process even in the presence of nonnative
hydrophobic interactions. As revealed from the free-energy
analysis that the disordered system was more readily affected by
nonnative hydrophobic interactions (Figure 1), the binding rate of
the disordered system showed greater amplitude of change
(Figure 5B). For the disordered system, the largest binding rate
was 1.46 times as large as that when nonnative hydrophobic
interactions were not included (KHP=0.0). However, for the
ordered system, the largest binding rate was only 1.08 times as
large as that at KHP=0.0. This indicates that the nonnative
molecular recognition
hydrophobic interactions further amplify the kinetic advantages of
IDPs in the binding process. The most striking finding was that the
strength of the nonnative hydrophobic interactions corresponding
to the maximum binding rate (KHPmax-rate) showed a strong
dependence on the chain flexibility (Figures 5A, C). The
disordered system exhibited a smaller KHPmax-ratethan the ordered
system. The reduction in the binding rate under strong nonnative
hydrophobic interactions was caused by non-specific kinetic traps
along the binding trajectory. Figure 5D exemplifies a binding
trajectory of a system with SQf(free)T=0.46 under KHP=1.50.
Non-specific intermediate states are clearly shown. The chain
flexibility dependence of KHPmax-rateindicates that, during the
binding process, IDPs are more ready to form non-specific binding
intermediates and even kinetically trapped misbinding states.
The encounter complex is stabilized
The kinetic advantage of IDPs originates from the chain
flexibility facilitating the encounter complex to evolve into the final
binding complex rather than escape to the unbound state [62]. To
investigate the influence of nonnative hydrophobic interactions on
such a mechanism, we made an analysis by dissecting the binding
process into a capture process and a further evolution process [62]:
pKIDzKIX/
?
kesc
kcap
pKID:::KIX ??
kevopKID:KIX,
where pKID+KIX is the unbound state, pKID?KIX is the native
bound state, while pKID???KIX is the loosely bound encounter
complex state formed by the capture event. We considered an
encounter complex occurred when the system evolved from an
unbound state to a state with Qb.0 (usually had one intermolec-
ular native contact). The effect of nonnative hydrophobic
Figure 1. Free-energy profiles of the binding process. Free-energy profiles were calculated using the fraction of native intermolecular contacts
(Qb) as a reaction coordinate for systems with different degrees of chain flexibility. (A–D) SQf(free)T=0.29, 0.46, 0.65, and 0.85 by tuning the
intramolecular interaction parameter a from 0.1, 1.0, 1.5 to 3.0. The two vertical dashed lines in panel (B) indicate the definition of the unbound state
(U), intermediate state (I), and bound state (B). The strength of the nonnative hydrophobic interactions (KHP) ranges from 0.00 to 1.50.
doi:10.1371/journal.pone.0015375.g001
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interactions on the capture rate kcap was rather small for all
systems and the disordered system possessed a slower capture rate
(Figure 6A). Unlike the capture process, the evolving and the
escape rates from the encounter state showed significant responses
to the presence of the nonnative hydrophobic interactions
(Figures 6B,C). Both Figures 6B and 6C show a two-stage profile,
i.e., an initial plateau stage followed by a sharp decrease stage. The
behaviors of the evolving and the escape rates as a function of the
nonnative hydrophobic interaction strength were synchronic, but
withopposite amplitude,namely,
(SQf(free)T=0.29) showed the greatest decrease of the evolving
the disordered system
rate and the smallest decrease of the escape rate, whereas the
ordered system (SQf(free)T=0.85) showed the smallest decrease of
the evolving rate and the greatest decrease of the escape rate.
Compared with Figure 5C, we also noticed that the points
corresponding to the sharp decreases of the evolving and the
escape rates were smaller than those corresponding to the sharp
decreases of the overall binding rate.
The above results showed that the effect of the nonnative
hydrophobic interactions on the binding process was primarily
exerted on the evolution and escape stages. Unlike electrostatic
interactions, which are long-range and accelerate the binding rate
by the steering effect [24], nonnative hydrophobic interactions are
short-range and their effects on the capture process is negligible
(Figure 6A). However, in the encounter state, nonnative
hydrophobic interactions contribute energetically to the stability
of the encounter complex and so a reduction in the evolving and
Figure 2. Thermodynamic properties of systems with different
degrees of chain flexibility. (A) Correlation between the population
of the intermediate state P(I) and the strength of the nonnative
hydrophobic interactions, KHP. (B) Effect of the nonnative hydrophobic
interactions on the stability of the complex which is measured by the
free-energy difference between the bound state and the unbound
state. (C) Effect of the nonnative hydrophobic interactions on the
transition temperature Tm.
doi:10.1371/journal.pone.0015375.g002
Figure 3. Properties of the free pKID domain in the presence of
nonnative hydrophobic interactions. (A) radius of gyration (Rg), (B)
average number of nonnative contacts (SNHP(free)T), and (C) the
average fraction of native contacts (SQf(free)T).
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escape rates were observed (Figures 6B,C). Evolution and escape
are two opposite processes in a binding event. Although increasing
the nonnative hydrophobic interactions reduces the escape rate, it
also reduces the evolving rate. Therefore, there is a balance
between the escape and evolving rates to lead to the maximum
binding rate.
Figure 4. Characterization of the number of nonnative contacts. (A–C) The average number of nonnative contacts SNHPT along the binding
process when the strength of nonnative hydrophobic interactions was increased: (A–C) KHP=0.50, 1.00, and 1.50. (D) Correlation between the
average number of nonnative contacts at the intermediate state SNHP(I)T and SQf(free)T. KHPwas set 1.50. (E) Correlation between the average
number of nonnative contacts in the bound state SNHP(B)T and SQf(free)T. KHPwas set 1.50. (F) Correlation between SNHP(I)T and KHP. The definitions
of the intermediate state and bound state are presented in Figure 1.
doi:10.1371/journal.pone.0015375.g004
Figure 5. Effect of the nonnative hydrophobic interactions on the binding kinetics. (A) Mean first passage time (MFPT) of the binding
process as a function of KHP. (B) Correlation between the relative binding rate and KHP. The relative binding rate was computed as
MFPT(KHP~0:0)=MFPT(KHP). (C) Correlation between the KHPcorresponding to the maximum binding rate, KHPmax-rate, and SQf(free)T. (D) A
typical binding trajectory for the system with SQf(free)T=0.46 under KHP=1.50.
doi:10.1371/journal.pone.0015375.g005
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