Controlling entropy to tune the functions of intrinsically ...€¦ · Controlling entropy to tune the functions of intrinsically disordered 1 regions Tilman Flock , Robert J Weatheritt1,

Controlling entropy to tune the functions of intrinsicallydisordered regionsTilman Flock1, Robert J Weatheritt1, Natasha S Latysheva andM Madan Babu

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ScienceDirect

Intrinsically disordered regions (IDRs) are fundamental units of

protein function and regulation. Despite their inability to form a

unique stable tertiary structure in isolation, many IDRs adopt a

defined conformation upon binding and achieve their function

through their interactions with other biomolecules. However, this

requirement for IDR functionality seems to be at odds with the

high entropic cost they must incur upon binding an interaction

partner. How is this seeming paradox resolved? While increasing

the enthalpy of binding is one approach to compensate for this

entropic cost, growing evidence suggests that inherent features

of IDRs, for instance repeating linear motifs, minimise the

entropic cost of binding. Moreover, this control of entropic cost

can be carefully modulated by a range of regulatory

mechanisms, such as alternative splicing and post-translational

modifications, which enable allosteric communication and

rheostat-like tuning of IDR function. In that sense, the high

entropic cost of IDR binding can be advantageous by providing

tunability to protein function. In addition to biological regulatory

mechanisms, modulation of entropy can also be controlled by

environmental factors, such as changes in temperature, redox-

potential and pH. These principles are extensively exploited by a

number of organisms, including pathogens. They can also be

utilised in bioengineering, synthetic biology and in

pharmaceutical applications such as increasing bioavailability of

protein therapeutics.

Addresses

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge

CB2 0QH, UK

Corresponding authors: Weatheritt, Robert J (rweather@mrc-

lmb.cam.ac.uk), Babu, M Madan ([email protected])1 These authors contributed equally to this work.

Current Opinion in Structural Biology 2014, 26:62–72

This review comes from a themed issue on Sequences and topology

Edited by L Aravind and Christine Orengo

http://dx.doi.org/10.1016/j.sbi.2014.05.007

0959-440X/# 2014 Elsevier Ltd. All rights reserved.

IntroductionThere is an increasing appreciation that structure is not an

essential prerequisite for many kinds of protein function

[1��]. An ever-growing number of studies have identified


extensive functional regions within polypeptide seg-

ments that do not form stable tertiary structure. These

regions, referred to as intrinsically disordered regions

(IDRs), exist in over 35% of human proteins [2,3] func-

tioning in all major cellular processes with significant

enrichment in signalling and regulation [4,5��]. Although

IDRs are capable of performing functions comparable to

structured regions, they vary significantly in their amino

acid composition [6] and their biophysical properties

[7��]. Together this suggests that IDRs are independent

units of protein function, capable of achieving biological

function in a manner that is distinct from that of struc-

tured regions [1��,7��].

Energy landscape of intrinsically disorderedregions in proteinsIn contrast to structured domains that are primarily gov-

erned by non-covalent tertiary contacts between second-

ary structural elements, IDRs do not adopt a well-defined

tertiary structure. Instead they are in a dynamic equi-

librium between different sets of conformational states

[8] (Figure 1). The probability of finding an IDR in a

certain state within this conformational (or statistical)

ensemble is related to its conformational free energy

landscape [7��,8]. The native state of IDRs is therefore

best described in terms of a relatively flat but rugged

energy landscape with numerous local energy minima

separated by low energetic barriers [9,10]. This strongly

contrasts with the energy landscape of a structured

domain, which exhibits a well-defined minimum energy

state [7��], resulting in one or a few clearly favoured

conformational states (Figure 1). Because of their flat

energy landscape, IDRs can exist in multiple energeti-

cally similar but conformationally different states. In

biophysical terms, this means that IDRs exhibit high

conformational entropy (Figure 1). When participating

in binding events with other biomolecules, IDRs must

pay an entropic cost due to transitioning from a large set of

conformations in the free form to a more restricted

arrangement in the bound form [11]. How do IDRs

exhibit functional properties when the potential entropic

penalty of engaging in functionality is likely to be high?

Recent studies have suggested that the large entropy loss

due to a disorder-to-order transition upon binding can be

counter-balanced by a comparable gain in enthalpy of

binding [12–14]. In many cases, this has been suggested

to result in an interaction that is characterised by high

specificity but low affinity of binding. IDRs achieve this

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Exploiting entropy for biomolecular interaction Flock et al. 63

Figure 1

Native Disordered State

Disordered

Many different conformations sampledSingle conformation dominates

Ordered

Fre

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Preferred Conformations Ordered State

Current Opinion in Structural Biology

The free energy landscape and conformational ensembles of IDRs can be dynamically tuned. The free energy landscape schematically represents any

possible conformation (x–y axes) a protein can adopt, as well as its respective free energy (z-axis) ( free energy landscape, upper row), and thus

describes the probability of finding any particular conformation in an ensemble of conformational states (statistical ensemble, lower row). Whereas

structured proteins have energy landscapes with a clearly preferred conformation (global minimum, right panel), disordered proteins in their native form

have a rugged, flat energy landscape allowing multiple conformations to be thermodynamically favourable and to be found in the statistical ensemble

(left panel). Several factors can modulate the free energy landscape of IDRs and restrict the statistical ensemble to fewer, more defined conformations

(middle panel). As conformational entropy decreases, so does the number of observed conformational states. In the most ordered state, the

disordered protein landscape turns into the funnel-shaped landscape of structured proteins, shifting the statistical conformational ensemble towards a

predominant conformation (right panel). This fine-tuning of the statistical ensemble of IDRs enables specific functions and tunability.

by having an extensive interaction interface capable of

participating in a large number of weak non-covalent

contacts [12,14]. However, there are many examples of

IDRs binding with a limited interaction surface area and/

or in their disordered state [15��,16]. So what other

additional mechanisms allow IDRs to overcome the

entropic penalty of binding and become functional? In

this article, we discuss the role of entropic contribution to

free energy (DG = DH � TDS; see Box 1) in IDR function

and highlight that minimising the relative entropic cost

upon binding is a prerequisite for functions mediated by

several IDRs. We further discuss that rather than con-

sidering the potentially large entropic cost incurred by


IDRs upon binding as a disadvantage, it may be regarded

as highly advantageous in many cases. This is because

modulation of the entropic cost enables IDRs to dyna-

mically change their energy landscapes. This results in

the creation of a wide range of regulatory functions that

can be actively and intricately tuned by intrinsic and

extrinsic factors.

How inherent features of IDRs permitexploitation of entropy for functionalityWhat are the potential ways to counter-balance the

entropic cost of binding of IDRs and modulate their

free energy landscape? Deriving from the definition of


64 Sequences and topology

Box 1 Thermodynamic properties in the context of polypeptides

and their ligands.

Relative enthalpy (DH) describes the change in internal energy of

the system as well as the impact on its surrounding environment. In

protein folding, this is mainly determined by electrostatic interactions

and non-covalent bonds between amino acids within secondary

structural elements of structured domains.

Relative entropy (DS) is a measure of the change in randomness

(number of distinct states) of a system. In biological systems, the main

factors contributing to entropy are the degree of conformational

freedom of the polypeptide chain, entropy of complex formation (e.g.

binding of ligand to the polypeptide), and entropy changes due to the

rearrangement of surrounding water (solvent) molecules.

Configurational entropy (DSconfig) of a protein is related to the

number of possible arrangements of a macromolecule. In statistical

mechanics terms, it refers to the number of microstates.

Conformational entropy (DSconform) of a protein is defined by the

degree of flexibility and the number of different conformations that can

be sampled. Although in some contexts used interchangeably, for

clarity we refer to the conventional definition of conformational

entropy as a subcategory of configurational entropy [97,98] to

account for other factors such as the number of combinatorial

configurations of ligand interactions in a system [25��].

Relative Free Energy (DG = DH � TDS) of a biomolecular interaction

describes the amount of work required (or energy released) to change

between different states (conformational states, or bound and

unbound form). DG < 0 will spontaneously shift an interaction towards

equilibrium.

Protein Free Energy Landscape describes the free energy of all the

possible conformational states a protein can adopt. The width of the

contour relates to the number of states whereas the depth relates to

the free energy of the state. In other words, every position on the

energy landscape represents the free energy of one particular con-

formational state (also see Figure 1).

Conformational (Statistical) Ensemble describes the set of all

possible conformations of a protein. The likelihood of finding a protein

in a particular conformational state is defined by the free energy

landscape. At equilibrium, the free energy landscape is related to the

probability of finding a certain state (as determined by the Boltzmann

relation).

Avidity describes the accumulated strength of the affinities of

multiple independent non-covalent binding in a biomolecular inter-

action.

free energy there are two possible approaches: either

increasing the gain of enthalpy of an interaction

(DH < 0) or minimising the entropic penalty incurred

upon binding (DS � 0). The strategy of increasing the

enthalpy of binding is typically achieved via large inter-

action interfaces of IDRs (Figure 2) and has been

reviewed extensively in recent years [12–14]. In this

section we will discuss how limiting the entropic penalty

that IDRs incur upon binding is an alternative approach

to achieve their function.

The most widely characterised IDR interaction interfaces

are linear motifs [17], which are normally less than 10

amino acids in length [5��,15��]. Linear motifs therefore

do not have extensive interfaces for maximising enthalpy

of binding. Instead, they seem to limit the entropic cost of

their interactions and maximise the efficient use of their

limited interface. This is achieved in several major

ways (see Figure 2b for examples). Firstly, the loss of


conformational entropy at the motif interface is limited.

Since many motifs do not form secondary structure

elements upon binding, the region mediating the

interaction remains flexible, permitting extensive back-

bone-mediated hydrogen bonds [18�,19] and thereby

maximising the potential enthalpy of binding within

the limited interface of the motif. When motifs do have

secondary structure, it is typically preformed and rigid in

the native state (e.g. polyproline stretches), minimising

the loss of conformational entropy upon binding [15��,20].

Secondly, the interaction partners of motifs tend to have

rigid interfaces, which ensure that upon binding, only the

motif-containing protein pays an entropic penalty [18�].Thirdly, in a large number of motif-mediated inter-

actions, water molecules act as bridging interfaces. By

having water molecules already bound in the free motif-

binding surface of the protein monomer, some of the

conformational entropic cost of binding is ‘prepaid’ by the

water molecule for the IDR-mediated interaction [18�].Finally, many motif interactions are further stabilised by

hydrophobic interactions [18�] that are driven by entropic

gain (due to the expulsion of water molecules). These

intrinsic biophysical properties of IDR-mediated inter-

actions have been finely tuned during evolution to med-

iate a diverse range of functionalities: from regulating co-

operative protein complex formation that determines

embryonic stem cell fate [21] to controlling cell division

by affecting protein degradation [22].

Because of the compact nature of motifs, many IDRs

contain multiple binding motifs (multivalent IDRs)

rather than just a single motif [15��]. These multivalent

IDRs are particularly prominent in processes such as

clathrin-mediated endocytosis [23] and T-cell receptor

signalling [24]. Importantly, the individual motifs within

the multivalent IDRs are able to act as independent

binding ‘hot spots’, allowing the regions linking the

motifs to remain flexible even in the bound state. Thus

multivalency limits the conformational entropic cost of

binding as only the motifs become rigid upon binding and

not the regions linking them (Figure 2). As a consequence

of the flexibility of the linker region, the IDR has a much

broader range of possible binding configurations in the

bound state due to the degeneracy in binding. This

property therefore contributes to a relative gain in con-

figurational entropy (see Box 1). This entropic gain

increases with the number of motifs (valency) in the

sequence [25��]. Thus, in multivalent IDRs, there is a

synergy between the relative gain in configurational

entropy by both (i) minimisation of conformational entro-

pic cost due to the linker regions still being flexible and

(ii) maximisation of the number of configurations allowed

due to the degeneracy in binding. Together, these attri-

butes result in an overall decrease in the free energy of

binding (DG) of the bound form (Figure 2). Furthermore,

with each additional binding motif, the overall avidity of

binding of the IDR, as well as the association time of the



Figure 2

• Repeating motifs in a multivalent IDRs show multiple configurations of binding in a complex resulting in a gain in configurational entropy

• Extensive interaction interfaces maximise enthalpy of binding

(b)

(a)

+ + +

Str

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xam

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The change in conformationalentropy upon binding can beminimised in two major ways:

Extensive interface with secondary structure

↑ interface area conform

Maximising enthalpy of binding

ΔG < 0: ΔH < 0 ΔG < 0: ΔS ≈ 0

ΔG = ΔH – T ΔS

Maximising enthalpy gain uponbinding (ΔHbinding<<0)

Minimising change in confor-mational entropy (ΔSconform)

Providing additional configurationalentropy (ΔSconfig) via repeated motifs

in multivalent IDRs

Minimising entropy loss upon binding

config

↑S

↑S

Motif interface without secondary structure

Intrinsically disordered region-binding domain

Intrinsically disordered region

• Linear motifs often do not require secondary structure formation for binding

• Multivalent IDRs have multiple motifs acting as binding “hot spots”, enabling their linker regions to stay flexible

• Nuclear localisation signals in proteins tagged for nuclear import remain disordered upon their binding to cognate importin proteins [93]

• Epsin-1 uses a multitude of motifs, punctuated by disordered linkers, to facilitate the formation of clathrin-coated invaginations during endocytosis [95]

• The natively unfolded N-terminal domain of Drosophila TAFII230 inhibits the DNA binding of TATA-box binding protein (TBP). TAFII230 folds upon binding and presents an extensive interaction surface [13]

• Interactions between the Src homology 3 (SH3) domain and its proline-rich motif (PRM) in the nephrin-NCK-N-WASP complex display combinatorial binding, resulting in phase transitions with eventual effects on actin nucleation [25]


Solutions to overcome the entropic cost of IDRs upon binding. (a) There are two major ways for tuning the free energy of IDRs (at constant temperature) to

overcome the entropic cost incurred upon binding: The entropic cost can be counter-balanced by increasing the gain of enthalpy of binding by forming

extensive interfaces (left panel) or by minimising the loss of entropy (middle and right panels). The latter can be achieved by either (middle panel) minimising

the conformational entropy upon binding by having multivalent interaction hot spots (motifs) allowing the linker regions between the motifs to remain flexible,

or by (right panel) allowing multiple configurations (binding modes) for binding the interacting partner, thereby resulting in a relative gain in configurational

entropy of the complex. (b) A table presenting examples for each strategy [13,25,93,95].

www.sciencedirect.com Current Opinion in Structural Biology 2014, 26:62–72


interaction complex increases [25��,26]. Similar bio-

physical benefits are also attributable to the ‘fuzzy bind-

ing’ of IDRs [16].

Thus, the conformational flexibility of IDRs can be

exploited to maximise their relative enthalpy gain and

minimise their relative entropy cost during binding. This

is enhanced in multivalent IDRs due to the presence of

independent binding sites. The intrinsic features of IDRs

may be therefore tinkered with during evolution to

exploit entropy to their advantage.

How extrinsic factors help IDRs exploitentropy for functionalityAnother way to deal with the entropic cost associated with

IDRs is to shift the statistical conformational ensemble

towards functional states via interactions with extrinsic

factors. These factors range from ligands [27] to post-

translational modifications (PTMs) [28] and from photons

[29] to protons [30]. In the following section, we will

discuss how these extrinsic factors act on IDRs to induce

folding and shift the conformational ensemble towards

functional states.

In biophysical terms, these extrinsic factors preferentially

interact and stabilise certain conformations of the stat-

istical ensemble [8]. Extrinsic factors can drive two inter-

dependent phenomena: induced folding and induced

unfolding. For induced unfolding, extrinsic factors pena-

lise the free energy of the preferred conformation, con-

verting the energy landscape from one with a defined

minimum to one that resembles more the flat, rugged

landscape of disordered regions. This mechanism is

exploited by chaperones when acidic conditions or high

temperature are encountered. Such situations can

denature and unfold certain regions of the chaperone,

thereby exposing functional sites [7��,30,31]. This may

enable refolding of the denatured client proteins by the

chaperone, as described for instance by an entropy trans-

fer model [32]. In contrast, induced folding comes at an

entropic cost, as the number of conformations that an IDR

can adopt is restricted in the folded state [12] (see

Figure 3). Once again, the extrinsic factor modulates

the energy landscape by providing an enthalpy gain to

the system, or by increasing the configurational entropy

(as discussed above). Conditional transition between the

two states (disordered and ordered) trigged by an extrinsic

factor allows a protein to switch between conformational

states, enabling diverse protein functions [33,34��,35].

The effects of induced folding of an IDR are not necess-

arily restricted to that particular local region and can

create a domino effect of conformational change in adja-

cent or distal regions of the polypeptide [36]. For

example, upon ligand binding a region could undergo a

disorder-to-order transition to expose a distal protein

interaction surface [28], a process often regulated by


changes in conformational entropy [37] (see Figure 3

for example). This ligand-induced alteration of protein

function at a distal site is defined as allostery [38] and has

been extensively studied in structured proteins [38]. Site-

to-site allosteric coupling often requires a physical con-

nection to transmit the conformational changes to a distal

site [39], for instance by an ‘allosteric wire’ [40] or a non-

covalent contact ‘pathway’ [41]. As IDRs in their native

state lack these types of rigid, ‘physical’ connections,

IDRs can instead modulate the conformational entropy

of the whole protein to induce allosteric coupling [42,43]

(Figure 3). This phenomenon relies on the fact that when

an allosteric ligand binds and induces folding in one part

of a protein, it will as a consequence restrict the possible

conformational ensembles of all adjacent regions [42,43].

This can increase (or decrease) the likelihood of folding of

an adjacent disordered region and therefore modulate its

function. This ‘allosteric ensemble’ based view means

that binding of an effector in one part of a protein can alter

the binding affinity of other regions despite there being

no direct physical non-covalent contact linking these two

regions [44��,45]. Taken together, these studies show that

the conformational change within IDRs can play an

important role in transmitting signals to distal sites within

proteins. This highlights the importance of dynamics of

IDRs in allosteric coupling [8,46] (see Figure 3).

Thus, the IDR energy landscape is highly susceptible

to external influences [7��], permitting the dynamic

switching between conformations [34��]. This allows

induced folding (as the extrinsic factor counter-bal-

ances the entropic cost of folding) as well as induced

unfolding. These basic principles can be extended to

achieve allosteric coupling in IDRs [42,47��], in such a

way that the same effector molecule can act on the

same protein either as an agonist or an antagonist

depending on which conformation is sampled (within

the energy landscape) by the IDR when encountering

the ligand [44��,48�].

Controlling entropy of IDRs facilitatestunability of protein functionAt first glance, the high intrinsic flexibility of IDRs

suggests that the large entropic cost of binding would

be a considerable obstacle to function. However, as dis-

cussed in the previous sections, this apparent disadvan-

tage can be exploited to create a mechanism to

incrementally tune protein function. Importantly,

because the relatively flat energy landscape of IDRs is

quite sensitive to local changes in the environment [7��],incremental changes in the environment allow a stepwise

modulation of the structural heterogeneity, or confor-

mational flexibility, of IDRs. In this manner, the proper-

ties of IDRs may serve to sense local changes in the

environment. In the following section, we describe

how this can be exploited to create novel and diverse

functionality.



Figure 3

• Oxidative stress unfolds bacterial chaperone Hsp33, enabling binding to multiple client proteins, counteracting aggregation and promoting refolding of the client protein [94]

• Conditional unfolding can be induced by extrinsic factors flattening the energy landscape resulting in a gain in conforamtional entropy

• Conditional folding can be induced by extrinsic factors shifting the energy landscape towards a defined minimum

• Protein-protein interfaces can be exposed upon ligand binding at a distal allosteric site inducing disorder-to-order transtions

• IDRs enable allosteric coupling between “domains”. Disorder-to-order transitions in one part of a protein reduce conformational entropy in adjacent regions thus increasing the likelihood of folding

• Phosphorylation of the RS domain within RNA-splicing SR proteins induces folding and enables protein interactions [28]

• The individual EF-hand domains of calmodulin become rigid upon calcium binding, allowing distal peptide binding [96]

• In the bacteriophage P1, binding of the disordered doc toxin to the intrinsically disordered C-terminal domain of phd protein induces folding of the adjacent N-terminal DNA-binding domain of phd by allosteric coupling [47]

• In the adenovirus early region 1A (E1A) oncoprotein, binding to the N-terminal region stabilises the adjacent domain, allowing it to form a ternary complex with the TAZ2 domain of the general transcription co-activator CBP and pRB (retinoblastoma protein) [44]

Strategy

No Ligands Bound

+

–

+

+

–

–

+

–

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Fre

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Fre

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First Ligand Bound Second Ligand Bound

(b)

(a)

Example


Extrinsic factors shift the energy landscape and conformational ensemble of IDRs permitting induced (un)folding, allosteric coupling and cooperative

binding. (a) Modulation of the free energy landscape and the conformational ensemble by ligands: (left panel) The flat, rugged, native free energy

landscape of a two-‘domain’ disordered protein results in an equal chance of observing each different conformations within a statistical ensemble

(middle panel). Binding of an allosteric ligand results in induced folding of the first ‘domain’, which restricts the conformational freedom of the second

‘domain’ which is still disordered. This shifts the statistical ensemble, increasing the likelihood of the occurrence of a folded conformation that can

interact with the ligand of the second ‘domain’ (right panel). This shift in the free energy landscape increases the probability that the second ligand can

interact with the second ‘domain’. This induced site-to-site allosteric interaction allows cooperative effects in proteins with disordered regions. The

ligands may range from small molecules to protein domains. (b) A table outlining strategies and examples of how extrinsic factors may modulate the

energy landscape of IDRs [28,44,47,94,96].

Tuning multivalent IDRs

Multivalent IDRs are especially suitable for tuning by

post-translational modifications (PTMs) and alternative

splicing, as individual motifs can be regulated indepen-

dently. PTMs in particular allow a tight and temporal


regulation of the binding sites in a disordered region.

Because of steric considerations and the potential to

mediate non-covalent interactions, an individual PTM

can switch a motif-mediated interaction on or off [49],

thereby shifting the statistical ensemble towards a



specific configuration. In particular, the combination of

multiple PTMs can allow stepwise modulation of func-

tion. For example, in kidney podocytes, the stepwise

phosphorylation of the motifs in the cytoplasmic tail of

the transmembrane protein nephrin modulates the

protein abundance of the nephrin-NCK-N-WASP system

required to stimulate the nucleation of actin filaments by

the Arp2/3 complex [25��]. Similar mechanisms are also

known to drive T-cell receptor signalling [50].

Alternative splicing (AS) is another powerful mechanism

known to regulate the valency of binding sites in a

disordered region and has been recently identified to

rewire protein-protein interactions [51,52] by modulating

motif-mediated interactions [53–55]. In particular,

alternative exons are enriched in protein segments coding

for recurring motifs [54], suggesting AS is a prominent

mechanism to tune multivalent assemblies. For example,

p73 isoforms with fewer repeating WW-domain binding

motifs form less stable complexes compared to the full-

length p73 isoform [56]. This difference reduces tran-

scriptional activity of the shorter isoforms [56]. Both

PTMs and AS can therefore incrementally modulate the

statistical ensemble of conformations, allowing rheostat-

like modulation of protein function in a cellular context.

In addition to the above regulatory processes, changes in

the local physical and chemical properties of the envi-

ronment can shift the energy landscape and reduce the

entropic cost required to form multivalent assemblies.

For example, the in vitro addition of biotinylated isoxa-

zole, a synthetic small molecule, acts as a template for a

spectrum of RNA-binding proteins containing intrinsi-

cally disordered low-complexity sequences to organise

into distinct RNA granules [57]. In another example,

oxidative stress and high temperatures in yeast induce

the formation of multivalent assemblies of Sup35, a

subunit of the translation termination complex with a

glutamine and asparagine-rich disordered N-terminus

[58]. This inhibits the function of Sup35, reducing the

reliability of translational termination and thereby affect-

ing translational efficiency. The increase in protein syn-

thesis errors has been suggested to expand the diversity of

the proteome [59] and thereby increase the probability

that at least a few individuals in a population will survive

the stressful conditions [60].

Taken together, these examples show that multiple bio-

logical, chemical, and physical factors can tune multi-

valent interactions involving IDRs. This can occur by

either (1) altering the number of available multivalent

motifs by using PTMs and/or alternative splicing to

modulate the configurational entropy gain and the overall

avidity of assembly formation, or by (2) changing the

entropic cost of the individual motif interactions using for

example environmental stimuli. This makes multivalent

IDR-mediated interactions a highly tunable system and


sensitive to a wide range of signalling inputs and environ-

mental cues.

Tuning induced (un)folding and allosteric coupling

In a manner similar to multivalent motif binding, induced

folding/unfolding and especially allosteric coupling be-

tween protein segments can be readily tuned by control-

ling the entropic cost required for (un)folding.

Phosphorylation is a prominent example due to its ability

to both contribute enthalpy, via ionic interactions, and

reduce conformational entropy by inducing local hydro-

gen bond formation resulting in the steric hindrance of

adjacent regions of the IDR. For example, RS (Arginine-

Serine rich) regions are IDRs found in SR proteins that

are involved in RNA splicing [28]. Phosphorylation of the

RS region induces folding and is a key activation step for

SR proteins [28]. Another regulatory mechanism is the

use of alternative promoters or different translation start

sites that tend to encode coding regions enriched in IDRs

[54,55]. The presence of disordered segments in DNA

binding proteins such as transcription factors can alter

DNA sequence specificity and affinity [51,53,61]. For

example, the three isoforms of glucocorticoid receptor

differ in the length of their disordered N-terminal region

due to different translation start sites [62]. This creates

isoforms with different transcriptional activities despite

having identical DNA-binding domains. The difference

in activity emerges because the different N-terminal

regions of the isoforms have different allosteric coupling

effects on the same DNA-binding domain [62]. This in

turn modulates the fraction of DNA-binding domains in

the correct conformation to activate transcription and

therefore the overall transcriptional activity of glucocor-

ticoid receptors in the system [62] (see Figure 3).

The power and versatility of tuning the function of IDRs

is especially apparent when considering the impact of

environmental factors on modulating the entropic cost of

protein-protein interactions. Modulation of the redox

potential is exploited in chloroplasts to regulate CP12,

an 80 amino acid intrinsically disordered protein (IDP)

[29]. In the presence of light, when sufficient reducing

equivalents and ATP are produced by photosynthesis, all

four cysteines in CP12 are reduced and the protein

remains in a disordered, unfolded state [29,63]. In the

dark however, the reducing equivalents are scarce, allow-

ing CP12 to form two disulphide bridges facilitating the

formation of a partially structured conformation [64]. In

this conformation, CP12 is able to form a ternary complex

with two enzymes critical for CO2 assimilation, effec-

tively blocking their activities [63] and thus causing the

inactivation of the Calvin cycle in the dark [29]. In

another example, the decreased pH in the cis-Golgi

promotes unfolding of the C-terminal of ER-resident

protein 44 (Erp44), permitting its recognition of other

incorrectly folded proteins and facilitating their traffick-

ing back to the ER [65].



An intriguing example of environmental differences act-

ing as an extrinsic control of conformational entropy is

associated with a disordered loop in a protein that is

important for sporulation in Neurospora crassa. This dis-

ordered segment modulates the protein aggregation prop-

erties of a fungal hydrophobin EAS [66��]. In solution,

EAS is monomeric due to the large conformational

entropy of the disordered loop, which prevents self-

assembly. However at the air-water interface, EAS rapidly

self-assembles as the hydrophilic-hydrophobic boundary

reduces the conformational flexibility of the loop. The

disordered loop can therefore no longer prevent self-

assembly [66��,67]. This is an elegant mechanism to

ensure fungal spores are only released at the air-water

interface and not underwater.

The above examples highlight how diverse biological

systems take advantage of conformational and configura-

tional flexibility of IDRs to incrementally tune a wide

range of highly malleable protein functions. The fact that

the energy landscape of IDRs can be dynamically modu-

lated by different factors to shift the conformational

ensemble towards particular states means that IDRs have

the potential to mediate diverse functions that are easily

regulated by a range of ‘functional triggers’.

Pathogens exploiting IDR tunability

The presence of such a malleable system can be co-opted

and exploitedbypathogens tohijack the regulatoryproteins

and processes of the cell [68–70]. For example, the enter-

ohaemorrhagic E. coli effector protein EspFU hijacks actin

polymerisation to attach to intestine walls using extensive

motif mimicry: the repetitive N-WASP-binding motifs of

EspFU synergistically activate actin assembly, an ability

which is enhanced as multivalency increases [71]. In a

similar mechanism, vaccinia virus actin tail nucleator A36

can associate with the AP2 adaptor complex via multiple

endocytic sorting motifs to promote actin polymerisation,

which facilitates viral particle fusions and therefore viral

spread [72]. The extensive use of multiple binding motifs

(multivalent IDRs) in viral proteins in a combinatorial

manner suggests that this is a prevalent strategy in mediat-

ing host-virus interactions [73,74].

In a similar manner, pathogens co-opt allosteric effectors,

for example in diphtheria toxin expression, which is

allosterically regulated by the repressor protein DtxR

in response to iron availability [75]. A large domain of

DtxR is molten-globule-like in solution, with metal bind-

ing triggering a disorder-to-order transition. This permits

binding of DtxR to the tox operator and repression of

transcription of the toxin [75]. In the absence of iron,

DNA-binding of DtxR is abolished and diphtheria toxin

is produced. The result is that the production of

diphtheria toxin is linked to the availability of iron, a

common signal for virulence factor expression in intra-

cellular microbial pathogens [75].


Translating knowledge into synthetic biology

The efficiency with which nature has been able to exploit

IDRs by modulating entropy to tune function suggests

the same principles can also be used effectively in syn-

thetic biology. The use of multivalent motifs has already

found a number of successful applications. In metabolic

engineering, SH3 and PDZ-binding motifs in particular

have been used to create highly tunable, multivalent

scaffolds capable of co-localising enzymes and increasing

local intermediate product concentrations [76,77] as well

as in creating customised cell-signalling pathways

[78�,79]. Repeating motifs within IDRs have also been

used to prolong the release of bioactive drugs into circu-

lation. Soluble fusion proteins with peptides interspersed

with protease scission motifs form a depot that can release

the active peptides upon contact with a natively occurring

protease [80]. Similarly, the fusion of proteins with long

disordered segments has been shown to increase solubi-

lity [67] and increase the bioavailability of protein thera-

peutics in the blood plasma [81]. Thus, controlling

entropy to tune the conformational ensemble of IDRs

enables scientists and bioengineers to understand, manip-

ulate, and exploit natural systems for regulating proper-

ties such as enzymatic activity, bioavailability and protein

solubility.

ConclusionsA number of recent studies have highlighted the key role of

controlling entropy of IDRs for modulating protein inter-

actions, folding and function [11,37,82–84]. In this article,

we have discussed emerging ideas suggesting that the high

entropic cost of IDRs is particularly susceptible to the

incremental modulation of IDR function by regulatory

mechanisms and environmental factors. This permits

extensive tuning of protein function in a manner analogous

to a rheostat. This is exemplified in multivalent IDR

interactions, induced (un)folding and allosteric coupling

of IDRs. We therefore suggest that the concept of modul-

ating the entropy of IDRs is a general principle underlying

the functional plasticity of such protein segments.

An important implication of this concept is that sequence

variation among orthologs can be tolerated as long as all

potential functional conformations remain energetically

accessible. Furthermore, the ability of IDRs to sample a

diverse range of functional conformational states might be

a key factor in enabling proteins to rapidly adapt to novel

environments. An appreciation of protein dynamics, con-

formational energy landscapes and functional plasticity

[85] could therefore be crucial to fully understand protein

evolution, especially of polypeptide segments that are

intrinsically disordered.

Advances in single molecule experiments [86], hydrogen-

deuterium exchange mass spectroscopy experiments [87],

and theoretical studies on sequence-structure ensembles

[88,89�] are expanding our knowledge of the regulation of



IDRs. In addition, recent developments combining NMR

techniques, such as residual dipolar coupling (RDC), with

molecular dynamic simulations [90,91] have enabled the

quantification of the entropic contribution to biomolecu-

lar interactions [11,37,82–84]. The application of these

techniques should direct future research towards under-

standing the tunability of IDR conformations, functions

and their regulation. Together these techniques might

allow a greater understanding of, and enable us to engineer,

higher order structures [92] and multivalent motif-

mediated interactions [5��]. This will in turn help increase

our understanding of the role of IDRs in hub proteins

and the central role of IDRs in signalling and regulation.

AcknowledgementsWe thank C Ravarani, B Lang, M Babor, G Murshudov and S Balaji fordiscussions and their comments on this work, as well as Lesly McKeanefrom the MRC-LMB visual aids department for assistance with figuredesign. This work was supported by the Medical Research Council(U105185859), HFSP (RGY0073/2010; MMB), the EMBO YoungInvestigator Program (MMB), ERASysBio+ (GRAPPLE; RJW; MMB), aCanadian Institute of Health Research (CIHR) Postdoctoral Fellowship(RJW) and the Boehringer Ingelheim Fonds (BIF; TF). We apologise to thecolleagues whose work was not cited owing to space constraints.

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Controlling entropy to tune the functions of intrinsically ...€¦ · Controlling entropy to tune the functions of intrinsically disordered 1 regions Tilman Flock , Robert J Weatheritt1,