Controlling Entropy to Tune the Functions of Intrinsically Disordered

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Controlling entropy to tune the functions of intrinsically

disordered regions

1 1

Tilman Flock , Robert J Weatheritt , Natasha S Latysheva and

M Madan Babu

Intrinsically disordered regions (IDRs) are fundamental units of extensive functional regions within polypeptide seg-

protein function and regulation. Despite their inability to form a ments that do not form stable tertiary structure. These

unique stable tertiary structure in isolation, many IDRs adopt a regions, referred to as intrinsically disordered regions

deﬁned conformation upon binding and achieve their function (IDRs), exist in over 35% of human proteins [2,3] func-

through their interactions with other biomolecules. However, this tioning in all major cellular processes with signiﬁcant

requirement for IDR functionality seems to be at odds with the enrichment in signalling and regulation [4,5 ]. Although

high entropic cost they must incur upon binding an interaction IDRs are capable of performing functions comparable to

partner. How is this seeming paradox resolved? While increasing structured regions, they vary signiﬁcantly in their amino

the enthalpy of binding is one approach to compensate for this acid composition [6] and their biophysical properties

entropic cost, growing evidence suggests that inherent features [7 ]. Together this suggests that IDRs are independent

of IDRs, for instance repeating linear motifs, minimise the units of protein function, capable of achieving biological

entropic cost of binding. Moreover, this control of entropic cost function in a manner that is distinct from that of struc-

can be carefully modulated by a range of regulatory tured regions [1 ,7 ].

mechanisms, such as alternative splicing and post-translational

modiﬁcations, which enable allosteric communication and

Energy landscape of intrinsically disordered

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

regions in proteins

entropic cost of IDR binding can be advantageous by providing

In contrast to structured domains that are primarily gov-

tunability to protein function. In addition to biological regulatory

erned by non-covalent tertiary contacts between second-

mechanisms, modulation of entropy can also be controlled by

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

environmental factors, such as changes in temperature, redox-

tertiary structure. Instead they are in a dynamic equi-

potential and pH. These principles are extensively exploited by a

librium between different sets of conformational states

number of organisms, including pathogens. They can also be

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

utilised in bioengineering, synthetic biology and in

certain state within this conformational (or statistical)

pharmaceutical applications such as increasing bioavailability of

ensemble is related to its conformational free energy

protein therapeutics.

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

Addresses best described in terms of a relatively ﬂat but rugged

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge

energy landscape with numerous local energy minima

CB2 0QH, UK

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

contrasts with the energy landscape of a structured

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

lmb.cam.ac.uk), Babu, M Madan ([email protected]) domain, which exhibits a well-deﬁned minimum energy

These authors contributed equally to this work.

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

conformational states (Figure 1). Because of their ﬂat

Current Opinion in Structural Biology 2014, 26:62–72 energy landscape, IDRs can exist in multiple energeti-

cally similar but conformationally different states. In

This review comes from a themed issue on Sequences and topology

biophysical terms, this means that IDRs exhibit high

Edited by L Aravind and Christine Orengo

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

http://dx.doi.org/10.1016/j.sbi.2014.05.007 conformations in the free form to a more restricted

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

Introduction counter-balanced by a comparable gain in enthalpy of

There is an increasing appreciation that structure is not an binding [12–14]. In many cases, this has been suggested

essential prerequisite for many kinds of protein function to result in an interaction that is characterised by high

[1 ]. An ever-growing number of studies have identified 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 Preferred Conformations Ordered State Free Energy Free Energy Free Energy Free Energy Landscape

Ordered Disordered Statistical Ensemble

Single conformation dominates Many different conformations sampled

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 IDRs upon binding as a disadvantage, it may be regarded

participating in a large number of weak non-covalent as highly advantageous in many cases. This is because

contacts [12,14]. However, there are many examples of modulation of the entropic cost enables IDRs to dyna-

IDRs binding with a limited interaction surface area and/ mically change their energy landscapes. This results in

or in their disordered state [15 ,16]. So what other the creation of a wide range of regulatory functions that

additional mechanisms allow IDRs to overcome the can be actively and intricately tuned by intrinsic and

entropic penalty of binding and become functional? In extrinsic factors.

this article, we discuss the role of entropic contribution to

free energy (DG = DH À TDS; see Box 1) in IDR function How inherent features of IDRs permit

and highlight that minimising the relative entropic cost exploitation of entropy for functionality

upon binding is a prerequisite for functions mediated by What are the potential ways to counter-balance the

several IDRs. We further discuss that rather than con- entropic cost of binding of IDRs and modulate their

sidering the potentially large entropic cost incurred by free energy landscape? Deriving from the deﬁnition of

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64 Sequences and topology

Box 1 Thermodynamic properties in the context of polypeptides

conformational entropy at the motif interface is limited.

and their ligands.

Since many motifs do not form secondary structure

Relative enthalpy (DH) describes the change in internal energy of elements upon binding, the region mediating the

the system as well as the impact on its surrounding environment. In interaction remains ﬂexible, permitting extensive back-

protein folding, this is mainly determined by electrostatic interactions

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

and non-covalent bonds between amino acids within secondary

maximising the potential enthalpy of binding within

structural elements of structured domains.

Relative entropy (DS) is a measure of the change in randomness the limited interface of the motif. When motifs do have

(number of distinct states) of a system. In biological systems, the main secondary structure, it is typically preformed and rigid in

factors contributing to entropy are the degree of conformational

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

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

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

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

Secondly, the interaction partners of motifs tend to have

rearrangement of surrounding water (solvent) molecules.

Conﬁgurational entropy (DSconﬁg) of a protein is related to the rigid interfaces, which ensure that upon binding, only the

number of possible arrangements of a macromolecule. In statistical motif-containing protein pays an entropic penalty [18 ].

mechanics terms, it refers to the number of microstates.

Thirdly, in a large number of motif-mediated inter-

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

actions, water molecules act as bridging interfaces. By

degree of ﬂexibility and the number of different conformations that can

be sampled. Although in some contexts used interchangeably, for having water molecules already bound in the free motif-

clarity we refer to the conventional deﬁnition of conformational binding surface of the protein monomer, some of the

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

conformational entropic cost of binding is ‘prepaid’ by the

account for other factors such as the number of combinatorial

water molecule for the IDR-mediated interaction [18 ].

conﬁgurations of ligand interactions in a system [25 ].

Finally, many motif interactions are further stabilised by

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

describes the amount of work required (or energy released) to change hydrophobic interactions [18 ] that are driven by entropic

between different states (conformational states, or bound and gain (due to the expulsion of water molecules). These

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

intrinsic biophysical properties of IDR-mediated inter-

equilibrium.

actions have been ﬁnely tuned during evolution to med-

Protein Free Energy Landscape describes the free energy of all the

iate a diverse range of functionalities: from regulating co-

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

contour relates to the number of states whereas the depth relates to operative protein complex formation that determines

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

embryonic stem cell fate [21] to controlling cell division

energy landscape represents the free energy of one particular con-

by affecting protein degradation [22].

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 Because of the compact nature of motifs, many IDRs

in a particular conformational state is deﬁned by the free energy contain multiple binding motifs (multivalent IDRs)

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

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

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

IDRs are particularly prominent in processes such as

relation).

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

Avidity describes the accumulated strength of the afﬁnities of

multiple independent non-covalent binding in a biomolecular inter- signalling [24]. Importantly, the individual motifs within

action. 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

free energy there are two possible approaches: either multivalency limits the conformational entropic cost of

increasing the gain of enthalpy of an interaction binding as only the motifs become rigid upon binding and

(DH < 0) or minimising the entropic penalty incurred not the regions linking them (Figure 2). As a consequence

upon binding (DS 0). The strategy of increasing the of the ﬂexibility of the linker region, the IDR has a much

enthalpy of binding is typically achieved via large inter- broader range of possible binding conﬁgurations in the

action interfaces of IDRs (Figure 2) and has been bound state due to the degeneracy in binding. This

reviewed extensively in recent years [12–14]. In this property therefore contributes to a relative gain in con-

section we will discuss how limiting the entropic penalty ﬁgurational entropy (see Box 1). This entropic gain

that IDRs incur upon binding is an alternative approach increases with the number of motifs (valency) in the

to achieve their function. sequence [25 ]. Thus, in multivalent IDRs, there is a

synergy between the relative gain in conﬁgurational

The most widely characterised IDR interaction interfaces entropy by both (i) minimisation of conformational entro-

are linear motifs [17], which are normally less than 10 pic cost due to the linker regions still being ﬂexible and

amino acids in length [5 ,15 ]. Linear motifs therefore (ii) maximisation of the number of conﬁgurations allowed

do not have extensive interfaces for maximising enthalpy due to the degeneracy in binding. Together, these attri-

of binding. Instead, they seem to limit the entropic cost of butes result in an overall decrease in the free energy of

their interactions and maximise the efﬁcient use of their binding (DG) of the bound form (Figure 2). Furthermore,

limited interface. This is achieved in several major with each additional binding motif, the overall avidity of

ways (see Figure 2b for examples). Firstly, the loss of binding of the IDR, as well as the association time of the

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Exploiting entropy for biomolecular interaction Flock et al. 65

Figure 2

(a)

ΔG = ΔH – TΔS

ΔG < 0: ΔH < 0 ΔG < 0: ΔS ≈ 0

Maximising enthalpy of binding Minimising entropy loss upon binding

Providing additional configurational Maximising enthalpy gain upon Minimising change in confor- Δ Δ Δ entropy ( Sconfig) via repeated motifs

binding ( Hbinding<<0) mational entropy ( Sconform)

in multivalent IDRs

+ + +

↑S ↑ interface area conform ↑ S config Extensive interface with secondary structure Motif interface without secondary structure Intrinsically disordered region-binding domain Intrinsically disordered region

(b) • Extensive interaction interfaces The change in conformational • Repeating motifs in a multivalent IDRs maximise enthalpy of binding entropy upon binding can be show multiple configurations of binding minimised in two major ways: in a complex resulting in a gain in • Linear motifs often do not require configurational entropy secondary structure formation for binding

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

• The natively unfolded N-terminal • Nuclear localisation signals in • Interactions between the Src homology domain of Drosophila TAFII230 proteins tagged for nuclear import 3 (SH3) domain and its proline-rich motif inhibits the DNA binding of TATA-box remain disordered upon their (PRM) in the nephrin-NCK-N-WASP binding protein (TBP). TAFII230 folds binding to cognate importin complex display combinatorial binding, upon binding and presents an proteins [93] resulting in phase transitions with extensive interaction surface [13] eventual effects on actin nucleation [25] • Epsin-1 uses a multitude of motifs,

Example punctuated by disordered linkers, to facilitate the formation of clathrin-coated invaginations during endocytosis [95]

Current Opinion in Structural Biology

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].

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66 Sequences and topology

interaction complex increases [25 ,26]. Similar bio- changes in conformational entropy [37] (see Figure 3

physical beneﬁts are also attributable to the ‘fuzzy bind- for example). This ligand-induced alteration of protein

ing’ of IDRs [16]. function at a distal site is deﬁned as allostery [38] and has

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

Thus, the conformational ﬂexibility of IDRs can be to-site allosteric coupling often requires a physical con-

exploited to maximise their relative enthalpy gain and nection to transmit the conformational changes to a distal

minimise their relative entropy cost during binding. This site [39], for instance by an ‘allosteric wire’ [40] or a non-

is enhanced in multivalent IDRs due to the presence of covalent contact ‘pathway’ [41]. As IDRs in their native

independent binding sites. The intrinsic features of IDRs state lack these types of rigid, ‘physical’ connections,

may be therefore tinkered with during evolution to IDRs can instead modulate the conformational entropy

exploit entropy to their advantage. of the whole protein to induce allosteric coupling [42,43]

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

How extrinsic factors help IDRs exploit an allosteric ligand binds and induces folding in one part

entropy for functionality of a protein, it will as a consequence restrict the possible

Another way to deal with the entropic cost associated with conformational ensembles of all adjacent regions [42,43].

IDRs is to shift the statistical conformational ensemble This can increase (or decrease) the likelihood of folding of

towards functional states via interactions with extrinsic an adjacent disordered region and therefore modulate its

factors. These factors range from ligands [27] to post- function. This ‘allosteric ensemble’ based view means

translational modiﬁcations (PTMs) [28] and from photons that binding of an effector in one part of a protein can alter

[29] to protons [30]. In the following section, we will the binding afﬁnity of other regions despite there being

discuss how these extrinsic factors act on IDRs to induce no direct physical non-covalent contact linking these two

folding and shift the conformational ensemble towards regions [44 ,45]. Taken together, these studies show that

functional states. the conformational change within IDRs can play an

important role in transmitting signals to distal sites within

In biophysical terms, these extrinsic factors preferentially proteins. This highlights the importance of dynamics of

interact and stabilise certain conformations of the stat- IDRs in allosteric coupling [8,46] (see Figure 3).

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

dependent phenomena: induced folding and induced Thus, the IDR energy landscape is highly susceptible

unfolding. For induced unfolding, extrinsic factors pena- to external inﬂuences [7 ], permitting the dynamic

lise the free energy of the preferred conformation, con- switching between conformations [34 ]. This allows

verting the energy landscape from one with a deﬁned induced folding (as the extrinsic factor counter-bal-

minimum to one that resembles more the ﬂat, rugged ances the entropic cost of folding) as well as induced

landscape of disordered regions. This mechanism is unfolding. These basic principles can be extended to

exploited by chaperones when acidic conditions or high achieve allosteric coupling in IDRs [42,47 ], in such a

temperature are encountered. Such situations can way that the same effector molecule can act on the

denature and unfold certain regions of the chaperone, same protein either as an agonist or an antagonist

thereby exposing functional sites [7 ,30,31]. This may depending on which conformation is sampled (within

enable refolding of the denatured client proteins by the the energy landscape) by the IDR when encountering

chaperone, as described for instance by an entropy trans- the ligand [44 ,48 ].

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

entropic cost, as the number of conformations that an IDR Controlling entropy of IDRs facilitates

can adopt is restricted in the folded state [12] (see tunability of protein function

Figure 3). Once again, the extrinsic factor modulates At ﬁrst glance, the high intrinsic ﬂexibility of IDRs

the energy landscape by providing an enthalpy gain to suggests that the large entropic cost of binding would

the system, or by increasing the conﬁgurational entropy be a considerable obstacle to function. However, as dis-

(as discussed above). Conditional transition between the cussed in the previous sections, this apparent disadvan-

two states (disordered and ordered) trigged by an extrinsic tage can be exploited to create a mechanism to

factor allows a protein to switch between conformational incrementally tune protein function. Importantly,

states, enabling diverse protein functions [33,34 ,35]. because the relatively ﬂat energy landscape of IDRs is

quite sensitive to local changes in the environment [7 ],

The effects of induced folding of an IDR are not necess- incremental changes in the environment allow a stepwise

arily restricted to that particular local region and can modulation of the structural heterogeneity, or confor-

create a domino effect of conformational change in adja- mational ﬂexibility, of IDRs. In this manner, the proper-

cent or distal regions of the polypeptide [36]. For ties of IDRs may serve to sense local changes in the

example, upon ligand binding a region could undergo a environment. In the following section, we describe

disorder-to-order transition to expose a distal protein how this can be exploited to create novel and diverse

interaction surface [28], a process often regulated by functionality.

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Exploiting entropy for biomolecular interaction Flock et al. 67

Figure 3

(a) No Ligands Bound First Ligand Bound Second Ligand Bound

+ +

– – Free Energy Free Energy Free Energy Free Energy Landscape

+ +

– – Conformational Ensemble

(b) Strategy Example

• Conditional unfolding can be induced by extrinsic factors • Oxidative stress unfolds bacterial chaperone Hsp33, enabling

flattening the energy landscape resulting in a gain in binding to multiple client proteins, counteracting aggregation and conforamtional entropy promoting refolding of the client protein [94]

• Conditional folding can be induced by extrinsic factors shifting • Phosphorylation of the RS domain within RNA-splicing SR the energy landscape towards a defined minimum proteins induces folding and enables protein interactions [28]

• Protein-protein interfaces can be exposed upon ligand binding • The individual EF-hand domains of calmodulin become rigid upon at a distal allosteric site inducing disorder-to-order transtions calcium binding, allowing distal peptide binding [96]

• IDRs enable allosteric coupling between “domains”. • In the bacteriophage P1, binding of the disordered doc toxin to the

Disorder-to-order transitions in one part of a protein reduce intrinsically disordered C-terminal domain of phd protein induces conformational entropy in adjacent regions thus increasing the folding of the adjacent N-terminal DNA-binding domain of phd by likelihood of folding 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]

Current Opinion in Structural Biology

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 regulation of the binding sites in a disordered region.

Multivalent IDRs are especially suitable for tuning by Because of steric considerations and the potential to

post-translational modiﬁcations (PTMs) and alternative mediate non-covalent interactions, an individual PTM

splicing, as individual motifs can be regulated indepen- can switch a motif-mediated interaction on or off [49],

dently. PTMs in particular allow a tight and temporal thereby shifting the statistical ensemble towards a

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68 Sequences and topology

speciﬁc conﬁguration. In particular, the combination of sensitive to a wide range of signalling inputs and environ-

multiple PTMs can allow stepwise modulation of func- mental cues.

tion. For example, in kidney podocytes, the stepwise

phosphorylation of the motifs in the cytoplasmic tail of Tuning induced (un)folding and allosteric coupling

the transmembrane protein nephrin modulates the In a manner similar to multivalent motif binding, induced

protein abundance of the nephrin-NCK-N-WASP system folding/unfolding and especially allosteric coupling be-

required to stimulate the nucleation of actin ﬁlaments by tween protein segments can be readily tuned by control-

the Arp2/3 complex [25 ]. Similar mechanisms are also ling the entropic cost required for (un)folding.

known to drive T-cell receptor signalling [50]. Phosphorylation is a prominent example due to its ability

to both contribute enthalpy, via ionic interactions, and

Alternative splicing (AS) is another powerful mechanism reduce conformational entropy by inducing local hydro-

known to regulate the valency of binding sites in a gen bond formation resulting in the steric hindrance of

disordered region and has been recently identiﬁed to adjacent regions of the IDR. For example, RS (Arginine-

rewire protein-protein interactions [51,52] by modulating Serine rich) regions are IDRs found in SR proteins that

motif-mediated interactions [53–55]. In particular, are involved in RNA splicing [28]. Phosphorylation of the

alternative exons are enriched in protein segments coding RS region induces folding and is a key activation step for

for recurring motifs [54], suggesting AS is a prominent SR proteins [28]. Another regulatory mechanism is the

mechanism to tune multivalent assemblies. For example, use of alternative promoters or different translation start

p73 isoforms with fewer repeating WW-domain binding sites that tend to encode coding regions enriched in IDRs

motifs form less stable complexes compared to the full- [54,55]. The presence of disordered segments in DNA

length p73 isoform [56]. This difference reduces tran- binding proteins such as transcription factors can alter

scriptional activity of the shorter isoforms [56]. Both DNA sequence speciﬁcity and afﬁnity [51,53,61]. For

PTMs and AS can therefore incrementally modulate the example, the three isoforms of glucocorticoid receptor

statistical ensemble of conformations, allowing rheostat- differ in the length of their disordered N-terminal region

like modulation of protein function in a cellular context. due to different translation start sites [62]. This creates

isoforms with different transcriptional activities despite

In addition to the above regulatory processes, changes in having identical DNA-binding domains. The difference

the local physical and chemical properties of the envi- in activity emerges because the different N-terminal

ronment can shift the energy landscape and reduce the regions of the isoforms have different allosteric coupling

entropic cost required to form multivalent assemblies. effects on the same DNA-binding domain [62]. This in

For example, the in vitro addition of biotinylated isoxa- turn modulates the fraction of DNA-binding domains in

zole, a synthetic small molecule, acts as a template for a the correct conformation to activate transcription and

spectrum of RNA-binding proteins containing intrinsi- therefore the overall transcriptional activity of glucocor-

cally disordered low-complexity sequences to organise ticoid receptors in the system [62] (see Figure 3).

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

oxidative stress and high temperatures in yeast induce The power and versatility of tuning the function of IDRs

the formation of multivalent assemblies of Sup35, a is especially apparent when considering the impact of

subunit of the translation termination complex with a environmental factors on modulating the entropic cost of

glutamine and asparagine-rich disordered N-terminus protein-protein interactions. Modulation of the redox

[58]. This inhibits the function of Sup35, reducing the potential is exploited in chloroplasts to regulate CP12,

reliability of translational termination and thereby affect- an 80 amino acid intrinsically disordered protein (IDP)

ing translational efﬁciency. The increase in protein syn- [29]. In the presence of light, when sufﬁcient reducing

thesis errors has been suggested to expand the diversity of equivalents and ATP are produced by photosynthesis, all

the proteome [59] and thereby increase the probability four cysteines in CP12 are reduced and the protein

that at least a few individuals in a population will survive remains in a disordered, unfolded state [29,63]. In the

the stressful conditions [60]. dark however, the reducing equivalents are scarce, allow-

ing CP12 to form two disulphide bridges facilitating the

Taken together, these examples show that multiple bio- formation of a partially structured conformation [64]. In

logical, chemical, and physical factors can tune multi- this conformation, CP12 is able to form a ternary complex

valent interactions involving IDRs. This can occur by with two enzymes critical for CO2 assimilation, effec-

either (1) altering the number of available multivalent tively blocking their activities [63] and thus causing the

motifs by using PTMs and/or alternative splicing to inactivation of the Calvin cycle in the dark [29]. In

modulate the conﬁgurational entropy gain and the overall another example, the decreased pH in the cis-Golgi

avidity of assembly formation, or by (2) changing the promotes unfolding of the C-terminal of ER-resident

entropic cost of the individual motif interactions using for protein 44 (Erp44), permitting its recognition of other

example environmental stimuli. This makes multivalent incorrectly folded proteins and facilitating their trafﬁck-

IDR-mediated interactions a highly tunable system and ing back to the ER [65].

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Exploiting entropy for biomolecular interaction Flock et al. 69

An intriguing example of environmental differences act- Translating knowledge into synthetic biology

ing as an extrinsic control of conformational entropy is The efﬁciency with which nature has been able to exploit

associated with a disordered loop in a protein that is IDRs by modulating entropy to tune function suggests

important for sporulation in Neurospora crassa. This dis- the same principles can also be used effectively in syn-

ordered segment modulates the protein aggregation prop- thetic biology. The use of multivalent motifs has already

erties of a fungal hydrophobin EAS [66 ]. In solution, found a number of successful applications. In metabolic

EAS is monomeric due to the large conformational engineering, SH3 and PDZ-binding motifs in particular

entropy of the disordered loop, which prevents self- have been used to create highly tunable, multivalent

assembly. However at the air-water interface, EAS rapidly scaffolds capable of co-localising enzymes and increasing

self-assembles as the hydrophilic-hydrophobic boundary local intermediate product concentrations [76,77] as well

reduces the conformational ﬂexibility of the loop. The as in creating customised cell-signalling pathways

disordered loop can therefore no longer prevent self- [78 ,79]. Repeating motifs within IDRs have also been

assembly [66 ,67]. This is an elegant mechanism to used to prolong the release of bioactive drugs into circu-

ensure fungal spores are only released at the air-water lation. Soluble fusion proteins with peptides interspersed

interface and not underwater. with protease scission motifs form a depot that can release

the active peptides upon contact with a natively occurring

The above examples highlight how diverse biological protease [80]. Similarly, the fusion of proteins with long

systems take advantage of conformational and conﬁgura- disordered segments has been shown to increase solubi-

tional ﬂexibility of IDRs to incrementally tune a wide lity [67] and increase the bioavailability of protein thera-

range of highly malleable protein functions. The fact that peutics in the blood plasma [81]. Thus, controlling

the energy landscape of IDRs can be dynamically modu- entropy to tune the conformational ensemble of IDRs

lated by different factors to shift the conformational enables scientists and bioengineers to understand, manip-

ensemble towards particular states means that IDRs have ulate, and exploit natural systems for regulating proper-

the potential to mediate diverse functions that are easily ties such as enzymatic activity, bioavailability and protein

regulated by a range of ‘functional triggers’. solubility.

Pathogens exploiting IDR tunability Conclusions

The presence of such a malleable system can be co-opted A number of recent studies have highlighted the key role of

and exploitedbypathogenstohijack theregulatoryproteins controlling entropy of IDRs for modulating protein inter-

and processes of the cell [68–70]. For example, the enter- actions, folding and function [11,37,82–84]. In this article,

ohaemorrhagic E. coli effector protein EspFU hijacks actin we have discussed emerging ideas suggesting that the high

polymerisation to attach to intestine walls using extensive entropic cost of IDRs is particularly susceptible to the

motif mimicry: the repetitive N-WASP-binding motifs of incremental modulation of IDR function by regulatory

EspFU synergistically activate actin assembly, an ability mechanisms and environmental factors. This permits

which is enhanced as multivalency increases [71]. In a extensive tuning of protein function in a manner analogous

similar mechanism, vaccinia virus actin tail nucleator A36 to a rheostat. This is exempliﬁed in multivalent IDR

can associate with the AP2 adaptor complex via multiple interactions, induced (un)folding and allosteric coupling

endocytic sorting motifs to promote actin polymerisation, of IDRs. We therefore suggest that the concept of modul-

which facilitates viral particle fusions and therefore viral ating the entropy of IDRs is a general principle underlying

spread [72]. The extensive use of multiple binding motifs the functional plasticity of such protein segments.

(multivalent IDRs) in viral proteins in a combinatorial

manner suggests that this is a prevalent strategy in mediat- An important implication of this concept is that sequence

ing host-virus interactions [73,74]. variation among orthologs can be tolerated as long as all

potential functional conformations remain energetically

In a similar manner, pathogens co-opt allosteric effectors, accessible. Furthermore, the ability of IDRs to sample a

for example in diphtheria toxin expression, which is diverse range of functional conformational states might be

allosterically regulated by the repressor protein DtxR a key factor in enabling proteins to rapidly adapt to novel

in response to iron availability [75]. A large domain of environments. An appreciation of protein dynamics, con-

DtxR is molten-globule-like in solution, with metal bind- formational energy landscapes and functional plasticity

ing triggering a disorder-to-order transition. This permits [85] could therefore be crucial to fully understand protein

binding of DtxR to the tox operator and repression of evolution, especially of polypeptide segments that are

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

DNA-binding of DtxR is abolished and diphtheria toxin

is produced. The result is that the production of Advances in single molecule experiments [86], hydrogen-

diphtheria toxin is linked to the availability of iron, a deuterium exchange mass spectroscopy experiments [87],

common signal for virulence factor expression in intra- and theoretical studies on sequence-structure ensembles

cellular microbial pathogens [75]. [88,89 ] are expanding our knowledge of the regulation of

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

70 Sequences and topology

8. Boehr DD, Nussinov R, Wright PE: The role of dynamic

IDRs. In addition, recent developments combining NMR

conformational ensembles in biomolecular recognition. Nat

techniques, such as residual dipolar coupling (RDC), with Chem Biol 2009:5789-5796.

molecular dynamic simulations [90,91] have enabled the

9. Fenwick RB, Esteban-Martin S, Salvatella X: Understanding

quantiﬁcation of the entropic contribution to biomolecu- biomolecular motion, recognition, and allostery by use of

conformational ensembles. Eur Biophys J 2011:401339-401355.

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

techniques should direct future research towards under- 10. Whitford PC, Sanbonmatsu KY, Onuchic JN: Biomolecular

dynamics: order–disorder transitions and energy landscapes.

standing the tunability of IDR conformations, functions

Rep Prog Phys 2012:75076601.

and their regulation. Together these techniques might

11. Marlow MS, Dogan J, Frederick KK, Valentine KG, Wand AJ: The

allow a greater understanding of, and enable us to engineer, role of conformational entropy in molecular recognition by

calmodulin. Nat Chem Biol 2010:6352-6358.

higher order structures [92] and multivalent motif-

mediated interactions [5 ]. This will in turn help increase 12. Wright PE, Dyson HJ: Intrinsically unstructured proteins: re-

assessing the protein structure–function paradigm. J Mol Biol

our understanding of the role of IDRs in hub proteins

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and the central role of IDRs in signalling and regulation.

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Acknowledgements

14. Zhou HX: Intrinsic disorder: signaling via highly speciﬁc but

We thank C Ravarani, B Lang, M Babor, G Murshudov and S Balaji for

short-lived association. Trends Biochem Sci 2012:3743-3748.

discussions and their comments on this work, as well as Lesly McKeane

from the MRC-LMB visual aids department for assistance with ﬁgure 15. Davey NE, Van Roey K, Weatheritt RJ, Toedt G, Uyar B,

design. This work was supported by the Medical Research Council Altenberg B, Budd A, Diella F, Dinkel H, Gibson TJ: Attributes of

(U105185859), HFSP (RGY0073/2010; MMB), the EMBO Young short linear motifs. Mol Biosyst 2012:8268-8281.

Investigator Program (MMB), ERASysBio+ (GRAPPLE; RJW; MMB), a This paper summarises features and biological implications of short linear

Canadian Institute of Health Research (CIHR) Postdoctoral Fellowship motifs that are frequently within disordered regions mediating protein–

(RJW) and the Boehringer Ingelheim Fonds (BIF; TF). We apologise to the protein interactions.

colleagues whose work was not cited owing to space constraints.

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