Protein plasticity driven by disorder and collapse governs the heterogeneous binding of CytR to DNA
Nucleic Acids Research
Protein plasticity driven by disorder and collapse governs the heterogeneous binding of CytR to DNA
Sneha Munshi 1
Soundhararajan Gopi 1
Sandhyaa Subramanian 1
Luis A. Campos 0
Athi N. Naganathan 1
0 National Biotechnology Center, Consejo Superior de Investigaciones Cient ́ıficas , Darwin 3, Campus de Cantoblanco, 28049 Madrid , Spain
1 Department of Biotechnology, Bhupat & Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras , Chennai 600036 , India
The amplitude of thermodynamic fluctuations in biological macromolecules determines their conformational behavior, dimensions, nature of phase transitions and effectively their specificity and affinity, thus contributing to fine-tuned molecular recognition. Unique among large-scale conformational changes in proteins are temperature-induced collapse transitions in intrinsically disordered proteins (IDPs). Here, we show that CytR DNA-binding domain, an IDP that folds on binding DNA, undergoes a coil-toglobule transition with temperature in the absence of DNA while exhibiting energetically decoupled local and global structural rearrangements, and maximal thermodynamic fluctuations at the optimal bacterial growth temperature. The collapse is shown to be a continuous transition through a combination of statistical-mechanical modeling and all-atom implicit solvent simulations. Surprisingly, CytR binds single-site cognate DNA with negative cooperativity, described by Hill coefficients less than one, resulting in a graded binding response. We show that heterogeneity arising from varying binding-competent CytR conformations or orientations at the singlemolecular level contributes to negative binding cooperativity at the level of bulk measurements due to the conflicting requirements of collapse transition, large fluctuations and folding-upon-binding. Our work reports strong evidence for functionally driven thermodynamic fluctuations in determining the extent of collapse and disorder with implications in protein search efficiency of target DNA sites and regulation.
Thermodynamic fluctuations of macromolecules are one of
the primary factors driving transcription, binding reactions,
allostery and thus the cellular responses to varied
environmental conditions (
). Macromolecules are therefore
highly sensitive to temperature modulations with the extent
of protein oligomerization, degradation and protein
disorder all dependent on temperature (
) and slaved to solvent
composition, structure––extent and strength of hydrogen
bonds in the bulk versus the first shell––and motions (10).
It is also now well established that thermodynamic stability
is a continuum ranging from intrinsically disordered (IDPs)
to well-folded rigid proteins; experimentally, the measured
stability (with reference to the folded state) can be
negative (i.e. only unfolded like conformations are populated) to
10–15 RT. The large conformational heterogeneity of IDPs
contributes to moonlighting functions (
), ‘fly-casting’ effects (16), structural changes
driven by post-translational modifications (
), fuzzy complexes (
) and many more (
In systems that are reasonably well-folded, changes in
thermal fluctuations at lower temperatures (<320 K) can
modulate the population, reconfiguration dynamics within
and kinetics between partially structured, intermediate and
even the unfolded states. On the other hand, unfolded or
disordered proteins collapse with temperature (
irrespective of their sequence composition that can range from
glycine-serine or poly-glutamine repeats (
) to proteins rich in hydrophilic residues (
One of the many questions that has kindled the interest on
disordered proteins is the effect of changing solvent
conditions on the dimensions of disordered polypeptide chains
to test for various polymer scaling laws that relate radius of
gyration (RG) to protein length (N), i.e. RG = ρ0 Nν where
ν is the Flory exponent (
). The exponent ranges from
1/3 for compact conformations (poor solvent), 1/2 for
point conditions (ideal- or random walk chain) to 3/5 for
expanded coils (or self-avoiding random walk chains in
good solvent) (
). Detailed experiments on multiple
disordered proteins suggest that under native-like conditions
protein chains are close to the -state where chain–chain
and chain–solvent interactions are effectively balanced (32).
The discussion above raises interesting questions. Are
disordered or unfolded protein collapse transitions first- or
second-order-like? Since disordered systems exhibit no
tertiary packing interactions and little secondary structure,
if any, it has been proposed that IDPs undergo
secondorder-like transitions or continuous or downhill
), i.e. the free energy landscape of such systems
are titled towards unfolded like conformations without any
macroscopic free energy barrier separating the varied
conformations. If disordered polymeric chains indeed undergo
a second-order like collapse transition as expected from
the unimodal FRET efficiency histograms, they should
exhibit thermodynamically decoupled structural transitions,
as shown for folded proteins with weak energetic coupling
), that has not been explored before for any IDP. At
critical or collapse transition points, the free energy
surfaces are flat in continuous transitions contributing to large
thermodynamic fluctuations. Accordingly, there should be a
near one-to-one correspondence between -state and
The near-universal observation of collapse in disordered
systems further raises the question of whether nature
finetuned the extent of conformational heterogeneity in
disordered proteins for modulating the binding affinity to their
partner ligands. If so, the large fluctuations implicit under
such conditions might result in non-trivial functional
output with potential promiscuous binding, a feature that is
increasingly being observed in disordered systems. We explore
these issues in the current work by studying the monomeric
and helical DNA-binding domain of the prokaryotic
protein CytR (cytidine repressor) that regulates the expression
of proteins responsible for nucleoside recycling (
is a unique system in that CytR is disordered (Figure 1A)
despite exhibiting high sequence complexity and folds upon
binding to its target sequence (udp promoter), highlighting
an extreme case of disorder-to-order transition (40).
Through a combination of experimental multi-probe
spectroscopic studies, accurate heat capacity measurements,
a statistical model and all-atom implicit solvent simulations
we show strong evidence that CytR follows a
second-orderlike but non-specific collapse transition with temperature.
We then test whether chain collapse is a functionally driven
feature encoded in disordered proteins by probing the
binding ability of different conformational states of CytR to
its partner DNA. We identify that at the optimal
bacterial growth temperature, CytR has multiple conformational
states to choose from - expanded, collapsed, folded-like.
This frustration results in an array of binding competent
poses, some specific and some non-specific, thus translating
into a graded response of the binding signal.
MATERIALS AND METHODS
All experiments on CytR and PurR were recorded in
freshly prepared, filtered and degassed 20 mM sodium
phosphate buffer at pH 7.0 without or with urea, unless
mentioned otherwise. Protein solutions were routinely
filtered through 0.22 m syringe filters before every
experiment. Far-UV and near-UV circular dichroism (CD)
spectra were recorded in a Jasco J-815 spectropolarimeter
connected to a Peltier system at a concentration of ∼25 and
∼100 M, respectively. The urea melts in the range of 0 to
6–8 M urea were recorded at 298 K under the same buffer
conditions as above. The fluorescence experiments were
performed in a Chirascan Plus qCD instrument (Applied
Photophysics Ltd., UK) by exciting ∼26 M of CytR in 10 × 10
mm pathlength cuvette at 274 nm and collecting the
spectra between 280 and 400 nm. The quantum yields were
estimated employing NATA as a reference (0.13 at 298 K in
Dynamic light scattering (DLS)
The changes in dimensions of CytR with temperature were
monitored using a DynaPro-MS/X instrument (Protein
Solutions, Charlottesville, VA, USA) coupled to a Peltier
temperature controller. CytR samples at different
concentrations (0.45, 0.90 and 1.35 mg/ml) were first centrifuged at
10000g for 10 minutes and then filtered with a 0.1 m
filter (Anotop 10, 0.1 m from Whatman). 50 l samples
were loaded on to a cuvette and the scattered light intensity
was collected at 90◦ at different temperatures (from 283 to
333 K, every 10 K). The translational diffusion coefficients
(D) were determined from the time-series scattering data
using the DYNAMICS autocorrelation analysis software v.6
(Protein Solutions). To account for solution non-ideal
effects, the diffusion coefficients were measured at different
protein concentrations (C) and the resulting D values were
fit to an equation of the form D = D0 · (1 + a · C) where D0
is the intercept. The D0 and the D measured at the lowest
protein concentration were employed to estimate the
hydrodynamic radius (Rh) from the Stokes–Einstein relation and
the temperature-dependence of water viscosity.
Differential scanning calorimetry (DSC)
Heat capacity thermograms were recorded in a
MicroCal VP-Capillary DSC with an automated sample injector.
The instrument was thoroughly equilibrated with multiple
buffer scans following which CytR protein solutions
ranging from ∼110 to 52 M were scanned thrice at a scan rate
of 1 K/min. Buffer-buffer baselines were routinely recorded
before and after every protein scan to check for
instrumental baseline drifts. The absolute heat capacity was then
determined as described by Sanchez-Ruiz and coworkers (
Variable barrier (VB) model
The VB model analysis was performed employing the
absolute heat capacity thermogram of CytR as described in the
original work (
). The final parameters are: α = 1554.9
kJ mol−1; characteristic temperature, T0 = 291.7 K; β =
−173.7 kJ mol−1 (together with α it determines the shape
of the free energy profile at T0 in downhill profiles; it
represents the barrier height in a two-state-like system);
asymmetry factor, f = 0.535.
Protein–DNA binding experiments
Double stranded udpO (5 -ATTTATGCAACGCA-3 )
tagged with ALEXA532 at 5 end was purchased from
IBA Lifesciences (the binding site is highlighted in bold).
Experiments were performed in 50 mM sodium phosphate,
30 mM sodium chloride and 1 mM EDTA, pH 6.0 buffer.
A starting DNA concentration of 300 nM was titrated
with different concentrations of CytR ranging from ∼1
nm to 100 M; the resulting change in anisotropies were
monitored following a 5 minute equilibration at every
titration step by exciting the dye at 530 nm and collecting
the emission at 580 nm in a Chirascan Plus qCD
instrument (Applied Photophysics Ltd., UK) equipped with a
fluorescence polarization accessory.
Implicit solvent replica exchange MC (REMC) simulations
Simulations were performed using CAMPARI stand-alone
package, employing ABSINTH implicit solvent model with
OPLS charges (
). The protein was placed in a 100 A˚
spherical shell with 105 explicit excess ion-pairs to simulate
43 mM ionic strength conditions. REMC simulations were
carried out with 20 temperature replicas, ranging from 280
to 430 K, with exchange attempts every 20 000 steps
resulting in an average exchange probability of ∼0.4 across
temperatures. Simulations were carried out starting from the
randomized initial structure with temperature-dependent
dielectric constant and solvation free energies as before (
Each of the replicas was run for 6 × 107 steps in
parallel; snapshots were collected every 500 steps and the first
3 × 107 steps were discarded from further analysis.
Onedimensional free energy profiles and two-dimensional
surfaces were generated using the weighted histogram analysis
Dimensions of coil and collapsed globular states
To quantify the extent to which the overall dimensions of
CytR change with temperature, we measured the
hydrodynamic radius through non-invasive dynamic light scattering
(DLS) experiments that do not require fluorescent probes.
The DLS experiments were performed at different
concentrations of the protein at each temperature from which the
effective hydrodynamic radii were extracted (see Materials
and Methods). As expected from the crowded NMR
), the Rh of CytR is found to be ∼24 A˚ at the lowest
temperature with the dimensions matching the size-scaling
empirical relationship of Uversky et al. (UE; shaded regions
in Figure 1B) (
). Upon temperature increase the overall
Rh decreases reaching a value of ∼17 A˚ at 333 K with a
collapse temperature (Tc) of ∼312 K. Interestingly, the
collapsed globule-like state (UC) of CytR exhibits a
hydrodynamic radius similar to that of the folded protein in the
presence of its cognate DNA (∼15 A˚; green triangle in Figure
1B). The small difference in Rh (∼12%) between the fully
collapsed state and the folded state is in agreement with the
relative collapsed state dimensions observed upon dilution
of denaturants in single-molecule FRET measurements on
folded proteins (
Thermodynamically decoupled conformational changes
Most disordered proteins exhibit little or no secondary
structure. Moreover, as the temperature is increased the
mean residue ellipticity at 222 nm generally becomes more
negative suggestive of higher secondary structure content
or more likely due to a decreased propensity for
polyproline like conformations (
). The disordered CytR, on the
other hand, displays secondary structure loss with
temperature (Figure 2A) in sharp contrast to the signal at 6 M urea.
As a reference, we studied the homologous DNA-binding
domain from PurR that exhibits a sequence similarity of
64% with CytR (identity of 48%); we find that PurR is well
folded (Figure 1A) and exhibits a sigmoidal-like unfolding
curve at the same experimental condition (Figure 2A). The
respective chemical denaturation induced unfolding profiles
at 298 K indicate a loss of secondary structure with
different apparent cooperativities expected of a disordered and
folded protein, respectively (Figure 2B). The comparison
highlights that disordered CytR populates helical-like
secondary structures at low temperature that are lost at higher
temperatures. It is also clear that the observed differences
originate from specific differences in the primary sequence
between the two proteins and not from the choice of
The sole tyrosine in CytR (Y53) provides another probe
to monitor the collapse transition. CytR at 6 M urea
reveals little changes in the near-UV CD signal at 280 nm.
CytR in the absence of urea, however, shows a weak
nearUV CD spectrum that further loses intensity with
increasing temperature mimicking far-UV CD observations
(Figure 2C). In both cases, the apparent midpoint of the collapse
transition appears a bit earlier thermodynamically
(compared to DLS) at ∼307 K when employing a two-state-like
equilibrium model between UE andUC. We also observe a
red shift in the fluorescence emission of tyrosine with
temperature, a phenomenon that is rarely reported in proteins;
this could be indicative of a temperature-driven transition
between sub-ensembles that populate folded-like to more
unfolded-like conformations. This in turn would have an
effect on the fluorescence emission of Y53 because of changes
in either its immediate tertiary environment (Figure 2C) or
through specific differences in nature and strength of
hydrogen bonding involving the tyrosyl group. Evidence for
this is observable in the fact that the red shifted emission is
dominant at even lower temperatures for CytR at 6 M urea
(i.e. when the ensemble is highly unfolded) while it
dominates the emission only at a higher temperature for CytR
at pH 7.0, 43 mM ionic strength conditions (Figure 2D and
Supplementary Figure S1). This structural change also
happens thermodynamically much earlier than either DLS or
CD with an apparent midpoint of ∼301 K (Figure 2E).
The observations above can be summarized in a
representation that highlights the variable transition midpoints from
different experimental probes in the range of
experimental temperatures (Figure 2F). Interestingly, significant
structural changes precede the actual collapse transition than
within the narrow range of ∼20 K monitored by DLS.
Thermodynamic fluctuations peak at the collapse transition midpoint
The large differences in the apparent collapse transition
temperatures are indicative of an energetically decoupled
system where different parts of the structure undergo
structural changes independent of one another upon
temperature modulations, reminiscent of a second-order-like
collapse transitions in homo-polymeric systems or proteins
in the globally downhill folding regime (
calorimetry experiments (DSC) provide a powerful avenue
to explore the nature of the collapse transition as they are
probe-independent and report on the overall
thermodynamics (47). Moreover, it is possible to extract the precise
nature of the collapse transition––specific or non-specific
and first- or second-order?––given the intrinsic connection
between the order of transition and the enthalpic
fluctuations reported by DSC (
). However, these experiments
are challenging to perform given the small excess heat
associated with structural transitions in disordered systems
and the necessity to generate absolute heat capacities that
requires multiple experiments at different protein
CytR is highly soluble (up till 300 M) and the
thermograms highly reversible enabling us to obtain precise
concentration-dependent apparent Cp (Figure 3A) from
which absolute heat capacities were extracted following the
protocol prescribed by Sanchez-Ruiz and coworkers (
The absolute heat capacity profile of CytR reveals no
pretransition region, no sharp excess heat capacity associated
with barrier-limited transition, and is well bounded by the
empirical folded Freire and unfolded Makhatadze-Privalov
(MP) baselines (Figure 3B) (
). This is, to the best of our
knowledge, the first absolute heat capacity measurement on
a disordered protein system. The unique features are
characteristic of a continuous second-order-like transition from
an unfolded but extended collection of conformations to a
collapsed state. Enthalpic fluctuations derived from the
excess heat capacities (i.e. after subtracting the native
baseline that includes contributions from non-conformational
fluctuations of the system) indicate that the collapsed
globule (UC) undergoes large amplitude enthalpic fluctuations
(even more than the UE) arguing against a specifically
collapsed state (dark green in Figure 3B). Moreover, the
fluctuations are maximal at the midpoint of the collapse
transition (∼312 K) indicating a system that is potentially
frustrated between multiple conflicting interactions. An
analysis of the thermogram employing the phenomenological
variable-barrier model (42), which is based on the Landau
theory of phase transitions, provides a quantitative picture
of the underlying thermodynamic landscape (see Materials
and Methods). The probability density continuously shifts
from low enthalpy to high enthalpy states with no evidence
for two macrostates coexisting at any condition thus ruling
out a barrier-limited two-state transition (Figure 3C).
DSC experiments therefore provide strong evidence for
a continuous or downhill-like collapse transition in CytR.
The maximal enthalpic fluctuations and hence the
maximal structural heterogeneity are observed at temperatures
∼310–313 K. Since the polymeric chain is expected to
exhibit no preferential intra-chain or chain-solvent
interactions under these conditions ( -state) (
), the dimensions
of CytR should match the expectation of an ideal chain.
To check for this, we convert the measured hydrodynamic
radius values to radius of gyration following a recent
) resulting in a change in RG from ∼26 A˚ in good
solvent conditions (low temperature) to ∼12 A˚ in poor
solvent (high temperature) (Supplementary Figure S2). At the
critical point of 312 K, the extracted RG value is ∼17 A˚ ,
close to the expectation for a polymer in its -state (∼17.9
A˚ ) from RG = 2.2N1/2 (in A˚ units and for N = 66) (
A flat landscape with multiple conformational sub-states from all-atom simulations
To explore the nature of transition further and to
identify the conformational characteristics of the ensembles
that are populated, we performed all-atom implicit solvent
replica-exchange Monte Carlo (REMC) simulations
employing the ABSINTH implicit solvent model (
includes specific temperature-dependent solvation
). Remarkably, the collapse transition midpoint and
the helical nature of the low-temperature ensemble are
semiquantitatively reproduced by the simulation methodology
without any modification to the default simulation
parameters (Figure 4A and B). Control simulations that do not
incorporate temperature-dependent solvation free energies
fail to capture the collapse transition in agreement with the
results of Schuler et al. (Supplementary Figure S3) (
Very little change is observed in the polar-apolar solvent
accessible surface area of the disordered ensemble on
temperature changes (Figure 4C). However, there are two
significant differences when compared to experiments: the
degree of predicted helical structure is much higher at low
temperatures and the amplitude of the collapse transition
is modest compared to that observed in experiments (30%
change). Given the agreement at the de-novo level, we
construct multiple 1D and 2D projections to extract the order
of the collapse transition. The underlying landscape is
expectedly rough but with minimal or zero thermodynamic
barrier separating the numerous states in all of the
projections including radius of gyration, number of native
contacts (Q; with the folded structure as a reference) and
backbone RMSD (Figure 4E–I, Supplementary Figure S4). This
observation is in line with the expectation of a
second-orderlike transition from current experiments, theoretical
predictions on CytR (
) and the unimodal probability
densities of end-to-end distance distributions from numerous
disordered proteins. At 310 K, a range of structures are
populated that include partial helical structure in all
native helices or a few of them (Figure 4J) similar to that
observed from a structure-based statistical mechanical model
). In addition to these, a collapsed sub-ensemble with
little secondary-structure is also evident (f in Figure 4I, J)
highlighting the complex phase space accessible under these
conditions; the population of this sub-ensemble increases
with temperature contributing to the collapsed and
compact globule (panel C in Supplementary Figure S5).
How is the collapsed state stabilized? We find here that
the fraction of native hydrogen bonds (those present in the
folded CytR structure) decrease with temperature while the
fraction of non-native hydrogen bonds increase with
temperature (Figure 4D). The latter primarily involves the
protein backbone and side-chain interactions involving the
uncharged polar amino acids of Asn, Gln, Ser and Thr. It
is important to note that these simulations have been
performed with a temperature-dependent solvation free energy
that has been shown to capture the collapse transition in
multiple proteins. Therefore, while solvation could be a
major factor determining the collapse, the resulting collapsed
state seems to be energetically stabilized by multiple
nonnative hydrogen bonds. Given that CytR loses helical
structure with temperature, these observations immediately
highlight that the balance between the energetic terms
determining helical structure and the solvation terms need more
precise calibration for better agreement with experiments.
The full-length CytR (that includes the DNA-binding and
oligomerization domain) is dimeric and highly promiscuous
with the ability to bind octamer udp half-sites with multiple
inter-repeat spacing and orientations (
promiscuity has been attributed to the disordered nature of CytR
DNA-binding domain (40). Does the change in the
dimensions of CytR DNA-binding domain affect DNA-binding
ability? Binding studies with fluorescently labeled udp
halfsite reveal broad binding isotherms that do not saturate in
the temperature range of 278–308 K with the anisotropy
values continually increasing with CytR concentration
(circles in Figure 5A). Accordingly, a 1:1 equilibrium model
does not provide a statistically convincing fit to the data
(Supplementary Figure S6; a poor 1:1 fit is also observed
in the original CytR article (
We therefore resorted to the Hill binding equation (
extract the K1/2 values (midpoint of binding) and the Hill
coefficients (nH) that report on binding cooperativity:
f = K1n/H2 + [Cyt R]nH
where f is the fraction of ligand bound. It fits the binding
isotherms very well (lines in Figure 5A) with the midpoint
of binding shifting to the left with increasing temperatures
(∼23 M at 278 K to ∼9 M at 308 K, Figure 5B), which
is unusual given that either parabolic trends or
weakening affinities are generally observed with temperature. The
Hill coefficients are surprisingly less than 1 in this
temperature range, 0.60 ± 0.02 at 278 K to 0.69 ± 0.02 at 308
K, and marginally increase with temperature (Figure 5C).
A possible explanation for such ‘negative cooperativity’ is
when two or more CytR monomers bind the udp half-site.
However, NMR experiments, through which the structural
model of CytR was developed, clearly indicate that
binding is monomeric even up till a CytR concentration of 400
); this is four times higher than the maximal
concentration employed in our experiments (∼100 M;
Figure 5A), thus ruling out higher order effects.
Cooperativity effects from ensemble measurements and in single-ligand
binding are rare (though frequently reported in enzyme
)) and, to our knowledge, this is first such instance
reported for protein-DNA binding.
What feature determines the apparent negative
cooperativity in a 1:1 binding? It is important to note that the
Hill equation and the associated mechanistic interpretation
of the stoichiometry is valid only when the ligand is fully
folded or when the binding pose is invariant or well-defined
). Physically, Hill coefficients are the second moments
of binding energy distributions (
) and therefore in 1:1
binding scenarios nH < 1 represents binding heterogeneity.
Since CytR is disordered under these conditions, the
apparent negative cooperativity reported here should be a
consequence of the associated heterogeneity with multiple
conformations or orientations of the folded domain binding DNA,
with the NMR structure corresponding to dominant
binding mode. In this regard, it is interesting to note that single
molecule experiments on ion-dependent folding of P4–P6
RNA reveal that though the bulk Hill coefficient is close
to 1, the parent single-molecule binding isotherms exhibit a
large spread in folding-binding free energies (56). The
corollary is that bulk binding isotherm can be thought of as a
superimposition of multiple isotherms from single molecules
binding with varying affinities (i.e. binding heterogeneity),
the effective sum of which translates to a slow rise in
experimental anisotropy with the ligand concentration.
To extract the extent of binding heterogeneity that
manifests as nH < 1, we first assume that the experimental
binding free energy of CytR to DNA from individual molecules
is normally distributed (and as observed in single-molecule
)). We then modulate the standard
deviation ( ) of this distribution, simulate individual 1:1
binding isotherms picking binding free energies values from the
normal distribution, average them to extract an apparent
bulk binding isotherm and identify the width that best
reproduces the experimentally observed Hill coefficients and
K1/2 values. nH values distinguishable from 1 (<0.9 as
experimental errors also contribute to the variability) are
observed only when is >2–2.2 kJ mol−1 (Figure 5D) and are
challenging to identify as they are comparable to the
thermal energy. In the case of CytR, the experimental Hill
coefficients can be recovered (Supplementary Figure S7) when
the width of the underlying binding energy distributions are
substantially larger than thermal energy, ranging from ∼5.1
kJ mol−1 at 278 K to ∼4.5 kJ mol−1 at 308 K (Figure 5E and
Our results reveal that the observed complex binding
isotherms arise from intrinsic heterogeneity in binding that
in turn can be attributed to the conformational landscape of
CytR. What is the functional advantage? It is well known
that a dynamic equilibrium between non-specific and
specific binding modes, for ‘search’ and ‘recognition’,
respectively, needs to exist in DNA-binding proteins to enable
efficient search of target sites (
). Simulations reveal that
1D sliding motions on DNA, that dramatically increase the
identification of target sites (i.e. facilitated diffusion (58)),
tune the affinity/specificity of DNA binding (
increase with increasing disorder and degree of one-state-like
character (‘downhillness’) in the underlying conformational
). This intricate requirement is exacerbated in
CytR, as it not only collapses with temperature but also
folds upon binding DNA.
It is therefore tempting to speculate that the collapse
transition is functionally driven to enable access to
binding conformational pose(s) (akin to the ‘conformational
selection’ mechanism) through large thermodynamic
fluctuations within the flat downhill-like free energy landscape at
near the optimal bacterial growth temperature. This is
particularly evident in the plots of Rg versus Q derived from
implicit solvent simulations wherein CytR is shown to sample
a large array of conformations some of which are
foldedlike at 310 K (high Q and low Rg in sub-ensemble b in
Figure 4J). Such a feature will proportionately minimize
nonspecific binding and is consistent with our observations of
a correlation between an increase in binding affinity and
(marginal) decrease in binding heterogeneity with
temperature (Figure 5B and C). A similar observation has also been
made on Zinc-finger binding domains with an increased
binding affinity correlated with a population shift towards
the ‘recognition’ mode (
Temperature-induced coil to globule collapse transitions
in unfolded or intrinsically disordered proteins have
generally been studied for understanding the balance of energetic
terms determining folding, to optimize force-fields and to
explore the limits and applicability of various scaling laws
in polymer physics. Given that simulations and experiments
on disordered states almost always point to collapse with
temperature or changing solvent conditions, it is natural
to expect this feature has evolved for functional purposes.
In this work, we show that the phase space accessible to
CytR is highly heterogeneous and dependent on the
immediate solvent conditions: at low temperatures, the protein is
expanded and exhibits random-coil statistics while at high
temperatures it collapses to a globule exhibiting large
thermodynamic fluctuations but stabilized by non-native
hydrogen bonds. The CytR conformational ensemble at the
optimal bacterial growth temperature is precisely balanced
between both compact and coil-like conformations (i.e. the
state) thus exhibiting ideal-chain dimensions and maximal
enthalpic fluctuations for potential functional reasons. The
observed negative binding cooperativity to single-site DNA
is a direct consequence of the diversity in molecular
dimensions translating into varying binding affinities, similar to
that reported in the disordered region of the Notch
receptor protein (
It is also possible that CytR folds and binds with
multiple orientations, each pose exhibiting a different binding
affinity to the udp half-site. In fact, significant and diffuse
electrostatic frustration is observable in the DNA-binding
face of CytR (from the structure of the DNA-bound state,
Supplementary Figure S8); this feature potentially
highlights why CytR is disordered in the absence of DNA,
the role of DNA in determining the conformational
landscape of CytR and the possible reason for heterogeneous
binding through multiple electrostatically equivalent
orientations. Structurally, this hints that an array of binding
modes is accessible to CytR, some which are specific and
some non-specific, a characteristic feature of even
structured DNA-binding proteins (
), that is visibly
exaggerated due to a combination or conflicts from collapse,
large enthalpic fluctuations and DNA-driven folding. The
non-specific poses should enable CytR to bind even
random DNA sequences within the bacterial genome with the
degree of structural ordering driven purely by the extent of
similarity of the numerous DNA sequences with the
specific site. In other words, the conformational space
sampled by CytR could be determined by the DNA
sequencestructure to enable a precise balance between one- and
three-dimensional diffusive modes facilitating an effective
search of target sites.
Supplementary Data are available at NAR online.
We thank the FIST facility sponsored by the Department
of Science and Technology (DST), India at the Department
of Biotechnology, IITM for the high-end instrumentation.
Wellcome Trust/DBT India Alliance Intermediate
Fellowship [IA/I/15/1/501837 to A.N.N.]. Funding for open
access charge: Wellcome Trust/DBT India Alliance.
Conflict of interest statement. None declared.
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