The multifaceted roles of intrinsic disorder in protein complexes

FEBS Letters xxx (2015) xxx–xxx

journal homepage: www.FEBSLetters.org

Review

The multifaceted roles of intrinsic disorder in protein complexes Vladimir N. Uversky ⇑ Department of Molecular Medicine and USF Health Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, Tampa, FL, USA Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow Region, Russian Federation Department of Biology, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia

a r t i c l e

i n f o

Article history: Received 4 May 2015 Accepted 2 June 2015 Available online xxxx Edited by Wilhelm Just Keywords: Intrinsically disordered protein Intrinsically disordered protein region Protein–protein interaction Protein complex Scaffold Regulation Polyvalent interaction

a b s t r a c t Intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs) are important constituents of many protein complexes, playing various structural, functional, and regulatory roles. In such disorder-based protein complexes, functional disorder is used both internally (for assembly, movement, and functional regulation of the different parts of a given complex) and externally (for interactions of a complex with its external regulators). In complex assembly, IDPs/IDPRs serve as the molecular glue that cements complexes or as highly flexible scaffolds. Disorder defines the order of complex assembly and the ability of a protein to be involved in polyvalent interactions. It is at the heart of various binding mechanisms and interaction modes ascribed to IDPs. Disorder in protein complexes is related to multifarious applications of induced folding and induced functional unfolding, or defines the entropic chain activities, such as stochastic machines and binding rheostats. This review opens a FEBS Letters Special Issue on Dynamics, Flexibility, and Intrinsic Disorder in protein assemblies and represents a brief overview of intricate roles played by IDPs and IDPRs in various aspects of protein complexes. Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Recent years clearly showed that the universe of functional proteins includes ordered, partially ordered, and completely disordered species. The structure-less intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs) are commonly found in various proteomes [1–6], where they functionally complement ordered proteins and domains, typically playing important roles in cell signaling, as well as regulating and controlling various crucial biological processes [7–19]. IDPs/IDPRs are very promiscuous binders that are constantly involved in various interactions with diverse partners [20,21] and are known to play key roles in protein–protein interaction networks [13,22–26]. Since IDPs/IDPRs are structurally heterogeneous [7,27,28], their functions may arise from a specific disordered form, from inter-conversion between disordered forms, and from transitions between disordered to ordered or ordered to disordered states [16,17,29–31]. Furthermore, a template-dependent folding of some IDPs defines their ability to bind to multiple partners, gaining very different structures in the bound state [12,32], and thereby being able to possess unrelated, even opposite functions [33]. The ⇑ Address: Department of Molecular Medicine, University of South Florida, 12901 Bruce B. Downs Blvd. MDC07, Tampa, FL 33612, USA. E-mail address: [email protected]

multifaceted disorder defines the multifaceted functionality of IDPs and IDPRs, which can act as entropic chains (linkers, clocks, bristles), display sites (target sites for post-translational modifications), effectors (modulators of the functionality of partners), or scavengers (capturers and storages of small ligands) [33]. Furthermore, large multiprotein complexes also take advantage of intrinsic disorder, where IDPs/IDPRs often serve as assemblers by assisting assembly [33]. Intrinsic disorder plays a number of important roles in organization, maintenance, and control of protein complexes, ranging from transient signaling complexes to stable oligomers. In relation to protein complexes, there are two different types of functional disorder: internal, which is disorder used for assembly, movement, and functional regulation of the different parts a given complex, and external, whose major role is in defining the interaction of this complex with external regulators. Irrespective of this internal/external classification, intrinsic disorder has three global functional implications in protein complexes, playing various structural, functional, and regulatory roles. This idea is illustrated by Fig. 1 that represents an oversimplified scheme of the involvement of intrinsic disorder in assembly, function, and regulation of protein complexes (Fig. 1A), while Fig. 1B shows this involvement in more detail. Some of the features shown in this figure are discussed in sections below.

http://dx.doi.org/10.1016/j.febslet.2015.06.004 0014-5793/Ó 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Please cite this article in press as: Uversky, V.N. The multifaceted roles of intrinsic disorder in protein complexes. FEBS Lett. (2015), http://dx.doi.org/ 10.1016/j.febslet.2015.06.004

2

V.N. Uversky / FEBS Letters xxx (2015) xxx–xxx

with extended shapes and large interface areas are located, whereas the ordered proteins that from complexes in a three-state mechanism are found within the bottom-right corner corresponding to the more globular and compact protomers [36]. These two types of protomers are separated by a boundary line defined by the fact that the maxima of per-residue surface and interface areas for stable monomers lie around 80 Å2 [36]. Therefore, the per-residue surface area versus the per-residue interface area plot can be used for differentiation of two-state and three-state protein complexes with known 3-D structure (see Fig. 2B). The idea of large surface areas serving as identifiers of IDPs that fold at complex formation is further illustrated by Fig. 2C that represents the results of the computational ‘‘disassembly’’ of a eukaryotic ribosome [38]. It is clear that almost all of the individual ribosomal proteins do not have a simple globular structure (i.e., structure that defines the smallest accessible area), but they do possess very unusual shapes [38]. These peculiar mostly non-globular shapes indicate that many ribosomal proteins are involved in the formation of the two-state complexes. Very similar behavior was also emphasized for the nucleosome-forming core histones [39]. It is clear that the ability of IDPs/IDPRs to be involved in the binding-induced formation of the sophisticated highly intertwined structures, where different parts of a given IDP penetrate to binding pockets of different protomers, can be considered as a molecular glue or cement that becomes rigid once the complex forms and thereby serves as a crucial means for stable complex formation. 2.2. Disorder and binding chain reactions

Fig. 1. Various functional structural and regulatory roles of intrinsic disorder in protein complexes. (A) General overview. (B) Illustration of basic entropic activities and roles based on disorder-to-order and order-to-disorder transitions.

2. Roles of intrinsic disorder in assembly of protein complexes 2.1. Binding-induced folding of IDPs/IDPRs as molecular glue cementing protein complexes It is well-known that protein complexes can be formed following two-state or three-state mechanisms [34–36]. The two-state mode of the protein complex formation is related to the scenario where the protomers are disordered in their unbound forms and fold at the complex formation (see Fig. 2A, right side). In other words, protomers of the protein complexes that are formed via a two-state mechanism are intrinsically disordered in their uncomplexed form and clearly undergo the binding-induced folding at the complex formation [37]. On the contrary, in the three-state mechanism, the complex is formed from the independently folded individual chains (see Fig. 2A, left side) [34,35]. Obviously, many complexes cannot be formed by these two mechanisms alone, and in reality some mutual adjustment and co-folding are required for almost any complex formation. Comparative structural analysis of protein complexes formed via the two-state and three-state mechanisms revealed that their monomers possess very distinctive features, with the per-residue interface and surface areas of protomers that form the three-state oligomers being significantly smaller than those of the protomers that form the two-state complexes [36]. As a result, in the per-residue surface area versus the per-residue interface area plot, the two-state and three-state complexes occupy very different areas. Here, the IDPs forming the two-state complexes occupy a broad area in the top-right part of the plot where the protomers

IDP-based complex formation frequently involves at least partial folding of IDPR(s) into specific structures [16,17,29–31,37,40– 45], and the disorder-based interactions are characterized by adaptability, promiscuity, and ability of a given IDPR to fold differently upon binding to different targets [12,32]. Also, the ability of an IDP to partially fold at interaction with its binding partner(s) opens a possibility of the ‘‘binding chain reaction’’ mechanism based on the sequential generation of novel binding sites by partial folding of new disordered partners engaged in the consecutive interaction with the existing complex [37]. This model is illustrated by Fig. 3 which shows how interaction between proteins A and B induces structural changes in B or/and A, leading to the creation of new binding site(s) necessary for the additional interactions between A and B that leads to the strengthening of the AB complex. Alternatively, an activated AB⁄ complex is created, where a novel binding site is formed for interaction with a new partner C. At the next stage, some mutual rearrangements take place in the newly formed ABC complex, leading to the creation of new binding sites in the activated ABC⁄ complex that is now ready to interact with a new partner D. Obviously, the stepwise recognition and binding defines the timing and specific order of the assembly of some complexes, e.g., where C cannot interact with A until the AB complex is formed (see Fig. 3) [37]. This model can describe the stepwise directional assembly mechanism of large proteinaceous complexes, such as the Bardet–Biedl syndrome (BBS) protein complex BBSome containing seven BBS proteins (BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, and BBS9) [46], the intraflagellar transport complex [47], the mammalian 20S proteasome [48], and the 60S ribosomal subunit [49]. For example, careful mutational analysis revealed that the BBSome is formed sequentially and directionally, where the BBS7 interacts first with BBS2 and BBS9 to form the BBSome core that serves as an assembly intermediate, to which BBS1, BBS5, BBS8, and finally BBS4 are sequentially added [46]. In line with the stepwise directional assembly model are the observations on the disassembly of protein complexes that always occurs sequentially, in such a way that the least amount of buried

Please cite this article in press as: Uversky, V.N. The multifaceted roles of intrinsic disorder in protein complexes. FEBS Lett. (2015), http://dx.doi.org/ 10.1016/j.febslet.2015.06.004

V.N. Uversky / FEBS Letters xxx (2015) xxx–xxx

3

Fig. 2. Roles of intrinsic disorder in assembly of protein complexes. (A) Folding-based classification of protein complexes that can be formed in a three-state mechanism (a), where protein folding and binding happen as two independent and subsequent steps. Alternatively, some proteins are formed in a two-state manner (b), where folding and binding occur simultaneously. (B) Plot of per-residue surface versus per-residue interface areas. Surface and interface area are normalized by the number of residues in each chain for the ribosomal proteins that were estimated as described in [36]. Proteins of the 40S and 60S subunits are shown by red and blue circles, respectively. A boundary separating ordered and disordered complexes is shown as black dashed line. (C) Computational disassembly of the eukaryotic ribosome from the yeast Saccharomyces cerevisiae (PDB ID: 3U5C and 3U5E; [136]). Structure of the proteinaceous component of the ribosome is shown at the center of the plot as a large complex, and structures of the individual ribosomal proteins are positioned around this central complex. The figure clearly shows that there are almost no ribosomal proteins with simple globular shape, and many of them contain long protrusions or extensions.

interface area is exposed [50–52]. In other words, the disassociation of a complex follows a specific pathway where at each disassembly step, the smaller interfaces are broken, whereas the larger interfaces are preserved [53,54]. Curiously, this disassembly scenario suggests that the reverse process, the assembly of a complex, should be initiated by the formation of a sub-complex with the largest interface. Since the largest interface typically originates from the binding-induced folding of IDPRs [36] (see also Fig. 2), the first steps in the complex formation are inevitably associated with the interactions between the disordered monomers leading to the formation of the two-state multimers via binding-induced folding [37]. In other words, IDPs/IDPRs are at the core of the protein complex assembly.

2.3. Role of intrinsic disorder in the multiform binding mechanism The described above sequential binding mechanism, where the binding-induced folding of IDPs/IDPRs plays a central role in the assembly of protein complexes, is not the only way of how intrinsic disorder can be utilized in the stepwise complex formation. In fact, IDPRs are needed for the multiform binding mechanism, where the complex formation between an IDP and its ordered target leads to conformational changes in a target and to the opening of a hidden binding site [37,45]. Here, a protein complex is formed sequentially, in a stepwise manner, via the emergence of binding intermediates which leads to the structural rearrangements in a partner molecule accompanied by the exposure of ‘‘hidden’’ binding site,

Please cite this article in press as: Uversky, V.N. The multifaceted roles of intrinsic disorder in protein complexes. FEBS Lett. (2015), http://dx.doi.org/ 10.1016/j.febslet.2015.06.004

4

V.N. Uversky / FEBS Letters xxx (2015) xxx–xxx

with disjoint and spatially distant binding sites on the surface of the binding partner. Mechanistically, there are at least two different interaction modes that define the formation of such polyvalent complexes, namely semi-static and dynamic.

Fig. 3. Model of the binding chain reaction. Reproduced with permission from [37].

which are accessible to an IDP due to its structural flexibility [45]. Fig. 4 illustrates this model by showing two oversimplified scenarios of an IDP binding to an ordered partner with a ‘‘hidden’’ binding site. In the first case, formation of a binding intermediate defines a crucial pause needed while waiting for the opening of the originally closed binding site (Fig. 4A). In the second case, the complex formation involves two sequential binding intermediates (Fig. 4B), with the emergence of a second intermediate being required for stabilization of the open state of the partner that provides an access to the originally hidden binding site. Obviously, more complex mechanisms are possible as well. It is important to note that the aforementioned ‘‘hidden’’ binding in the ordered partner of an IDP site can be open either spontaneously or as a result of allosteric interaction [37]. 2.4. Intrinsic disorder and polyvalent interactions Quite often, disorder-based complexes are formed via the IDP(s) wrapping around binding partner(s) [20]. These are polyvalent complexes where several different segments of an IDP interact

Fig. 4. Two models illustrating multiform binding mechanism, where the interaction occurs between an IDP and an ordered partner with a ‘‘hidden’’ binding site. (A) A simple model of interaction with one binding intermediate. (B) A more complex model with two sequential binding intermediates. Reproduced with permission from [37].

2.4.1. Semi-static polivalent complexes In semi-static polyvalent complexes, the ordered segments of the wrapping IDPs are connected by the disordered regions, and the secondary structure elements of such wrappers possess hardly any intramolecular interactions, instead forming very intensive intermolecular contacts with a binding partner [20]. One of the illustrative examples of such semi-static polyvalent complex is given by protein phosphatase 1–inhibitor 2 complex (PP1-I2 complex) [55]. In this complex, I2 utilizes three disjoined regions, residues 12–17, 44–56, and 130–169, to simultaneously interact with three spatially distant PP1 sites, the regulator-binding region, the RVXF groove, and the substrate-binding cleft, respectively [55]. Although I2 is mostly disordered in the unbound form, its regions involved in interaction with PP1 gain well-defined structures upon binding. For example, in the PP1-I2 complex, the first I2 binding region has an irregular structure consisting of a mixture of hydrophobic associations and hydrogen bonds, the second region represents a short b-strand followed by a short a-helix, and the third region has two short a-helices connected by a sort linker region [55]. Importantly, more than half of I2 remains disordered even in the bound state, since segments 1–11, 18–43, 57–129, and 170–206 of I2 are not observed in the crystal structure of the PP1-I2-complex [55]. Other known examples of the semi-static polyvalent complexes include calpastatin–calpain [56], p27Kp1–cyclin-dependent kinase 2–cyclin A [57,58], and b-catenin–T cell factor [59]. Furthermore, many DNA- and RNA-interacting proteins are also flexible wrappers [20]. For example, numerous transcription factors, regulatory proteins, and other proteins that interact with DNA often contain multiple zinc finger motifs connected by flexible linkers and wrap around the DNA in a spiral manner [60]. Other known DNA wrappers are proteins with AT-hook motifs, such as the HMGA proteins. A typical AT-hook motif consists of a conserved and palindromic core Pro-Arg-Gly-Arg-Pro sequence that also includes a variable number of positively charged Lys and Arg residues on either side of the core sequence [61]. This AT-hook binds to the adenine– thymine (AT) rich DNA by adopting a crescent or hook-like structure around the minor groove of a target DNA [62]. HMGA proteins contain three AT-hooks, although some proteins contain as many as 30 [63]. Also, many ribosomal proteins are known to wrap over the rRNA surface [64–66]. 2.4.2. Fuzzy/dynamic polivalent complexes Another interesting example of semi-static bipartite complex is given by the interaction of eukaryotic initiation factor 4E (eIF4E) with eIF4E binding proteins (4E-BPs) which is crucial for the cap-dependent translation initiation [67]. It is known that three eukaryotic 4E-BPs and eIF4G share a YXXXXLF eIF4E-binding motif [68]. Although the ability of this motif to efficiently interact with eIF4E have been known for a long time, the binding of peptides containing the canonical binding site is significantly different from that of full-length 4E-BPs [69,70], with the affinity of full-length 4E-BPs being approximately two to three orders of magnitude higher than those of the peptides [70]. Recent NMR analysis revealed that 4E-BP2 is mostly disordered in solution but contains significant fluctuating secondary structure with a high helical propensity in the canonical binding site [67]. This protein can efficiently bind eIF4E using a bipartite mode of interaction involving the canonical 54YXXXXLF60 eIF4E-binding motif and a highly conserved 78IPGVT82 site [67]. The result of this interaction is a dynamic or fuzzy complex [71], in which 4E-BP2 maintains a

Please cite this article in press as: Uversky, V.N. The multifaceted roles of intrinsic disorder in protein complexes. FEBS Lett. (2015), http://dx.doi.org/ 10.1016/j.febslet.2015.06.004

V.N. Uversky / FEBS Letters xxx (2015) xxx–xxx

significant degree of disorder that can be described as an ensemble of conformations sampled even in its bound state [67]. This bipartite interaction of 4E-BP2 with eIF4E illustrates the ‘‘1 + 1 > 2’’ concept, since each of the two aforementioned binding elements of 4E-BP2 individually has submicromolar affinity, whereas the full-length 4E-BP2 forms the nanomolar complex with an eIF4E surface [67]. The dynamic nature of this complex is manifested by the ability of the 54YXXXXLF60 and 78IPGVT82 sites to efficiently exchange on and off of the eIF4E surface within the context of the very stable eIF4E-4E-BP2 complex [67]. 2.4.3. Dynamic polivalent complexes of shuffle or ‘‘hot potato’’ type An intriguing model of the highly dynamic shuffle complex has been recently proposed to describe the tri-partite interaction of the intrinsically disordered hNHE1cdt (which is the C-terminal distal tail, cdt, of the human Na+/H+ exchanger 1, hNHE1) with the extracellular-signal-regulated kinase-2 (ERK2) [72]. Based on the NMR analysis it has been concluded that the intrinsically disordered hNHE1cdt has the most NHE1 phosphorylation sites, contains three putative kinase recognition D-domains (D1–D3), and interacts directly with inactive (ia) ERK2 in a tri-partite Shuffle complex involving a D-domain, an F-site, and a substrate site [72]. The authors showed that the hNHE1 D-domains play a crucial role in the ERK2 activation and are involved in the regulation of the hNHE1 phosphorylation [72]. It has been also emphasized that in this hNHE1cdt-iaERK2 Shuffle complex, several binding sites of hNHE1cdt are involved in concomitant, non-cooperative, tri-partite interaction with iaERK2, where the hNHE1cdt sites do not affect each other and do not cooperate to increase the overall affinity, but ‘‘shuffle’’ dynamically, being sometimes off, sometimes on, thereby functioning similarly to holding a hot potato [72]. The authors emphasized that their model is based on the 40-years-old hot potato hypothesis proposed by Perham to describe channeling of substrates and intermediates in multi-enzyme complexes [73]. 2.4.4. Polivalent inhibitors Multivalent interactions involving the linked associations of ligands binding to multiple sites on a receptor confer increased affinity compared to binding to monovalent receptors [74]. The idea of cooperative polyvalent binding has been successfully exploited during the development of the polyvalent inhibitors of anthrax toxin produced by the causative agent of anthrax, Bacillus anthracis [75,76]. In its active form, this toxin represents a complex that consists of a heptamer formed by the membrane-bound PA63 fragment of the receptor-binding protein, protective antigen (PA), tightly bound to two bacterial enzymes, edema factor (EF, which is an adenylate cyclase that has an inhibitory effect on professional phagocytes) and lethal factor (LF, a protease acting specifically on macrophages, causing their death and the death of the host) [77,78]. Although several peptides-inhibitors have been found that can block the interaction of the heptameric PA63 with EF and LF, these inhibitors were shown to bind to their targets with low affinities [75,76]. However, when multiple copies of a peptide that binds weakly to the heptameric PA63 and prevents its interaction with enzymatic moieties where covalently linked to a flexible backbone, the resulting polymeric, polyvalent inhibitor was shown to prevent assembly of the toxin complex in vitro and blocked toxin action in an animal model [75,76]. 2.5. Different types of disorder-based protein complexes Disorder-based protein complexes are very diverse [20] and, depending on the disorder status of the bound constituents, can be classified as static or dynamic/fuzzy complexes. A multitude of binding modes attainable by IDPs has been systemized to form

5

a portrait gallery of disorder-based complexes, with some illustrative members of such assemblies being various static complexes such as surface-bound molecular recognition features, wrappers, chameleons, penetrators, huggers, intertwined strings, cylindrical containers, connectors, armature, tweezers and forceps, grabbers, tentacles, pullers, stackers of b-arcs, as well as diverse dynamic complexes, ranging from various fuzzy complexes to cloud contacts, proteins playing interaction staccato, and polyelectrostatic-type complexes [20]. Also, Fig. 1B shows that disorder-based protein assemblages can be permanent (e.g., scaffolds, stochastic machines, effectors, assemblers, etc.) or transient (such as display sites, annealers, chaperones, etc.) 3. Intrinsic disorder in functions and regulations of protein complexes Fig. 1 shows that in addition to crucial structural roles related to the assembly of disorder-based protein complexes, IDPs/IDPRs have numerous functional and regulatory activities in these complexes. Similar to structural roles, these functional/regulatory activities of intrinsic disorder can be manifested as a result of disorder-to-order or order-to-disorder transitions, or can have pure entropic nature. Many of the functions that IDPs/IDPRs play in protein complexes are similar to the functions traditionally ascribed to non-complexed IDPs, such as recognition, signaling, regulation, and control [7,10,13–17,21,30,31,33,79,80]. In fact, the disorder-based functional control of protein complexes utilizes IDPRs as specific means for recognition, regulation, activation, deactivation, and inhibition; as sites of various posttranslational modifications; and as an instrument for the controlled response to environmental changes. Since the topics of IDP functionality and control have been, and still are, a subject of numerous research articles and reviews, interested readers are addressed to the plethora of papers dedicated to this subject. The sections below cover some unique aspects related to the involvement of disorder in function and regulation of protein complexes. 3.1. Scaffolds and stochastic machines The major functions of scaffold proteins are the activities directed toward the orchestrating of the ‘‘enforced proximity’’, where proteins belonging to common and/or interlinked pathways are brought together by a specific scaffold protein that directs, coordinates, and regulates their action [81]. The abundance and roles of intrinsic disorder in members of this crucial functional class of proteins have been described in several articles [81–83]. It has been emphasized that scaffold proteins selectively bring together specific proteins within signaling pathways to facilitate and promote interactions between them, and, thereby, are intimately involved in temporal, spatial, orientational, and contextual aspects of the said signaling complex [82]. Among the enforced proximity-related activities of signaling scaffolds are various molecular mechanisms related to the use of intrinsic disorder in fly-casting, allosteric modification, structural isolation of partners, palindromic binding, ease of encounter complex formation, utilization of molecular recognition features, modulation of interactions between bound partners, masking of intramolecular interaction sites, protection of normally solvent-exposed sites, binding site overlap, maximized interaction surface per residue, toleration of high evolutionary rates, reduced constraints for alternative splicing, efficient regulation via posttranslational modification, efficient regulation via rapid degradation, and enhancing the plasticity of interaction and molecular crowding [82]. Recently, an important role of bivalent and polybivalent scaffolds in assembly of protein complexes has been emphasized

Please cite this article in press as: Uversky, V.N. The multifaceted roles of intrinsic disorder in protein complexes. FEBS Lett. (2015), http://dx.doi.org/ 10.1016/j.febslet.2015.06.004

6

V.N. Uversky / FEBS Letters xxx (2015) xxx–xxx

[74]. Here, two monovalent IDP chains are incorporated into a bivalent macromolecular scaffold, within which each disordered chain possesses one binding site. The two IDP chains involved in the formation of such a bivalent scaffold are linked via binding to a dimeric protein with two symmetrical binding sites or by self-association [74]. Curiously, the formation of bivalent scaffolds might result in a duplex with multiple additional bivalent sites, creating a polybivalent scaffold, and promote subsequent self-association and/or higher order organization of the IDP components [74]. The formation of a bivalent scaffolds are not necessarily accompanied by detectable changes in protein structure and dynamics [74]. Illustrative examples of bivalent and polybivalent scaffolds include complexes between Swallow and LC8, Nup159 and LC8, and the dynein intermediate and light chains [74]. 3.2. Protein complexes and dormant disorder Although the well-recognized and broadly accepted output of an IDP interaction with the binding partner is a disorder-to-order transition resulting in a (partial) folding of an IDP to more structured conformation [8,20,84–91], many IDPs/IDPRs remain predominantly disordered in the bound state, at least outside the binding interface [92–96]. It has been recently emphasized that functions of some ordered proteins rely on the decrease in the amount of their ordered structure [97]. In other words, these ordered proteins require local or even global functional unfolding, which has induced nature and transient character [97]. This conditional disorder [98], also known as cryptic or dormant, or transient disorder or functional induced unfolding [97], can be awoken by passive environmental factors that are not dependent on any specific interaction between the protein and its partners, and correspond to modification of some global parameters of the protein environment (e.g., changes in pH, temperature, the redox potential, mechanical force, or light exposure) and active factors that involve specific interaction of a protein with its environment and include interactions with membranes, ligands, other proteins, nucleic acids, or various posttranslational modifications, or the release of autoinhibition [97]. Some illustrative examples of interaction with other proteins that cause functional unfolding include the partial unfolding of BCL-xL induced by PUMA binding, unfolding of target proteins by ATP-dependent proteases and the mitochondrial import machinery, action of chaperones with the unfoldase activity, and transient unfolding of the flagellar protein during their export [97]. Another example is given by the calmodulin-induced activation of a vital phosphatase calcineurin, which is a heterodimer of a 19 kDa B chain and a 58–64 kDa A chain consisting of the catalytic domain, a B chain binding helix, a regulatory domain, autoinhibitory domain, and a C-terminal tail [99]. The regulatory domain of calcineurin represents a transiently disordered region, which, during activation, undergoes a series of conformational transitions from a folded state, to disordered form, and then back to folded conformation upon the formation of a final complex with the calmodulin via the ‘‘catch and release’’ mechanism [99]. 3.3. Entropic activities of IDPs/IDPRs in protein complexes 3.3.1. Stochastic machines An extreme case of an intricate role of intrinsic disorder in function of a highly dynamic signaling complex is given by so-called ‘‘stochastic machines’’ [100]. A founding member of this class of scaffold proteins is the axis inhibition (Axin) protein that colocalizes b-catenin, casein kinase Ia (CKI-a), and glycogen synthetase kinase 3b (GSK3b) to form a complex crucial for the Wnt signaling pathway [100]. Here, b-catenin, CKI-a, and GSK3b bind to distant sites of very long IDPR of Axin, yielding structured domains

connected by long flexible linkers [100–103]. It was proposed that this complex represents a ‘‘stochastic machine’’ (see Fig. 5) that works not by coordinated conformational changes, but by stochastic, uncoordinated movements of the linkers and their bound proteins enabling productive kinase-substrate collisions leading to phosphorylation [100]. It was also emphasized that there are hundreds of axin-like proteins in the human proteome, suggesting that the ‘‘stochastic machine’’ mechanism is likely to be exceedingly common, especially within the disorder-rich eukaryotic cell [100]. 3.3.2. Intrinsic disorder in binding rheostats IDP-based complexes can contain larger or smaller amounts of ordered structure. However, there are some complexes where almost no structure is formed in an IDP that binds to an ordered partner. An illustrative example of such interaction is given by the polyelectrostatic binding mode, describing the interaction of a highly charged IDP containing several binding motifs (cyclin-dependent kinase inhibitor Sic1 that is known to contain nine phosphorylation sites, each creating a sort linear binding motif for Cdc4, Cdc4 phospho-degron, CPD) with a folded globular partner, Cdc4, a substrate recognition subunit of a ubiquitin ligase containing the single primary binding pocket for CPD [104–107]. Based on NMR analysis it has been concluded Sic1–Cdc4 complex represents a highly dynamic ensemble where multiple CPDs exchange in and out of the binding pocket of Cdc4 in such a way that only directly interacting residues are transiently ordered, whereas the rest of Sic1 remains disordered [105]. Since the flexibility and disorder of phosphorylated Sic1 make all binding motifs equally accessible to Cdc4, all the CPDs of Sic1 are able to interact with Cdc4, one at a time, in a dynamic equilibrium. Weak individual affinities of these interactions permit the efficient exchange of bound CPDs. As a result of the fast interconversion between multiple flexible conformations, Sic1 does not present discrete charges to interact with Cdc4, but generates a mean electrostatic field that originates from the net charge of the whole IDP that helps unbound phosphates to contribute to the affinity via long-range electrostatic

Fig. 5. The stochastic machine mechanism. This figure shows a possible configuration for the complex involving Axin, b-catenin, GSC-3b, and CKI-a. Axin is shown with color variation to make its pathway easier to follow. The dashed line corresponds approximately to the location of the G295-A500 disordered segment [137]. Axin binds to CKI-a (at two separate sites), to GSK-3b, and also to b-catenin. Since the b-catenin binding site of Axin is located between the GSK-3b and CKI-a interaction sites, and since the two binding sites with CKI-a may lead to the formation of a loop, b-catenin becomes close to both kinases. Hence, the formation of this b-catenin destruction complex pulls all the proteins together, and substantially raises their local concentrations. Because the phosphorylation sites are in a disordered region of b-catenin and because the various binding sites are all in a long disordered region in Axin, random motions of these flexible regions can readily bring about the substrate–enzyme collisions needed for function. Reproduced with permission from [100].

Please cite this article in press as: Uversky, V.N. The multifaceted roles of intrinsic disorder in protein complexes. FEBS Lett. (2015), http://dx.doi.org/ 10.1016/j.febslet.2015.06.004

V.N. Uversky / FEBS Letters xxx (2015) xxx–xxx

interactions with the positively charged surface of Cdc4 [104]. Therefore, the highly dynamic Sic1–Cdc4 complex is formed via a cumulative electrostatic interaction of all Sic1 charges, regardless of whether they are actually bound to the binding site of the Cdc4 or not [106,107]. Intriguingly, the fact that Sic1 has many sub-optimal CPD motifs that act in concert to mediate Cdc4 binding defines the ability of Sic1 to serve as a binding rheostat, where the affinity of the Sic1–Cdc4 complex is directly proportional to the phosphorylation degree of Sic1, with efficient phosphorylation-dependent recognition by Cdc4 requiring a minimum of six of the nine CDK phosphorylation sites in Sic1 [108].

7

(SNAP-25) [128]. The members of this complex are differently distributed between the target plasma membrane and the vesicle membrane, where a binary complex t-SNAREs consisting of syntaxin and SNAP-25 is located on the target plasma membrane, whereas v-SNAREs (synaptobrevin, also known as VAMP2) is included in the vesicle membrane [129]. Individual t- and v-SNAREs are largely disordered and mediate membrane fusion via binding-induced folding, resulting in the formation of a stable zipper-like four-helix bundle that brings two membranes into close proximity for fusion [130–132]. 3.5. Intrinsic disorder and partner protection

3.3.3. IDPs/IDPRs and membrane-less organelles Recently, we hypothesized that IDPs and hybrid proteins with long IDPRs can serve as important drivers of the intracellular liquid–liquid phase separations that generate various membrane-less organelles [109]. Such membrane-less organelles are formed due to the colocalization of molecules at high concentrations within a small cellular micro-domain; they are devoid of membranes and are highly dynamic, with their components being involved in direct contact with the surrounding nucleoplasm or cytoplasm [110,111]. Some of the most known examples of these organelles are PML bodies or nuclear dots, or PODs [112], perinucleolar compartment [113], the Sam68 nuclear body [113], paraspeckles [114], nuclear speckles or interchromatin granule clusters [115], nucleoli [116], processing bodies [117], germline P granules [118,119], Cajal bodies [120], centrosomes [121], and stress granules [122]. It is believed that these cytoplasmic and nuclear membrane-less organelles are formed via a common mechanism related to the intracellular phase transitions [109,123], which may be triggered by changes in concentration of IDPs, changes in the concentrations of specific small molecules or salts, or changes in the pH and/or temperature of the solution, and can be further regulated by the various posttranslational modifications and alternative splicing of the phase-forming proteins, or by the binding of these proteins to some definite partners [109]. These phase transitions or liquid–liquid phase separations originate from the different effects of macromolecules on the structure and solvent properties of water, and are related to the high concentrations of macromolecular solutes [109], where at low concentrations of macromolecules the solution exists as a single phase, while at high concentrations, phase separation occurs [124]. The formation of the membrane-less organelles is not accompanied by significant structural changes, as it is often stabilized by patterned electrostatic interactions [125]. The appearance of a new liquid phase in these systems provides a distinct solvent environment for proteins, nucleic acids, and other solutes. Differences in solute–solvent interactions in the two phases commonly lead to unequal solute distribution. As a result, a new liquid phase may be specifically enriched or depleted in particular solutes [109]. In agreement with these considerations, nuage or germ granules found as phase-separated organelles in live cells and also formed via a phase transition of the disordered tails of Ddx4 in vitro were shown to provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA [125]. 3.4. Disorder-to-order transitions in SNARE complex and membrane fusion The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is a molecular engine that plays a crucial role in the vesicle fusion in eukaryotes [126,127]. In neurons, the ternary SNARE complex in neurons contains syntaxin, synaptobrevin, and synaptosome-associated protein of 25 kDa

Finally, an important role of IDPs in protein complexes is related to their ability to protect binding partners (see Fig. 1B). Some illustrative examples include: (i) RNA-binding proteins (which are often highly disordered) playing crucial roles in the prevention of genome instability by excluding formation of harmful RNA/DNA hybrids and by specific binding to mRNAs, nascent transcripts, non-coding RNAs, and damaged DNA [133]; (ii) intrinsically disordered cold-regulated (COR) proteins that protect chloroplast membranes during freezing through binding and folding [134]; and (iii) ubiquitin binding proteins Rhp23, Dph1 and Pus1 from fission yeast (all predicted to be mostly disordered) protecting multiubiquitin conjugates against deubiquitination [135]. Acknowledgment I am very thankful to Alexey Uversky for careful reading and editing of this manuscript. References [1] Dunker, A.K., Obradovic, Z., Romero, P., Garner, E.C. and Brown, C.J. (2000) Intrinsic protein disorder in complete genomes. Genome Inform. Ser. Workshop. Genome Inform. 11, 161–171. [2] Ward, J.J., Sodhi, J.S., McGuffin, L.J., Buxton, B.F. and Jones, D.T. (2004) Prediction and functional analysis of native disorder in proteins from the three kingdoms of life. J. Mol. Biol. 337, 635–645. [3] Uversky, V.N. (2010) The mysterious unfoldome: structureless, underappreciated, yet vital part of any given proteome. J. Biomed. Biotechnol. 2010, 568068. [4] Schad, E., Tompa, P. and Hegyi, H. (2011) The relationship between proteome size, structural disorder and organism complexity. Genome Biol. 12, R120. [5] Xue, B., Dunker, A.K. and Uversky, V.N. (2012) Orderly order in protein intrinsic disorder distribution: disorder in 3500 proteomes from viruses and the three domains of life. J. Biomol. Struct. Dyn. 30, 137–149. [6] Peng, Z. et al. (2015) Exceptionally abundant exceptions: comprehensive characterization of intrinsic disorder in all domains of life. Cell. Mol. Life Sci. 72, 137–151. [7] Dyson, H.J. and Wright, P.E. (2005) Intrinsically unstructured proteins and their functions. Nat. Rev. Mol. Cell Biol. 6, 197–208. [8] Dyson, H.J. and Wright, P.E. (2002) Coupling of folding and binding for unstructured proteins. Curr. Opin. Struct. Biol. 12, 54–60. [9] Wright, P.E. and Dyson, H.J. (1999) Intrinsically unstructured proteins: reassessing the protein structure–function paradigm. J. Mol. Biol. 293, 321–331. [10] Dunker, A.K., Silman, I., Uversky, V.N. and Sussman, J.L. (2008) Function and structure of inherently disordered proteins. Curr. Opin. Struct. Biol. 18, 756– 764. [11] Cortese, M.S., Uversky, V.N. and Dunker, A.K. (2008) Intrinsic disorder in scaffold proteins: getting more from less. Prog. Biophys. Mol. Biol. 98, 85– 106. [12] Oldfield, C.J., Meng, J., Yang, J.Y., Yang, M.Q., Uversky, V.N. and Dunker, A.K. (2008) Flexible nets: disorder and induced fit in the associations of p53 and 14-3-3 with their partners. BMC Genomics 9 (Suppl. 1), S1. [13] Uversky, V.N., Oldfield, C.J. and Dunker, A.K. (2005) Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling. J. Mol. Recogn. 18, 343–384. [14] Dunker, A.K., Brown, C.J., Lawson, J.D., Iakoucheva, L.M. and Obradovic, Z. (2002) Intrinsic disorder and protein function. Biochemistry 41, 6573–6582. [15] Dunker, A.K., Brown, C.J. and Obradovic, Z. (2002) Identification and functions of usefully disordered proteins. Adv. Protein Chem. 62, 25–49. [16] Dunker, A.K. et al. (2001) Intrinsically disordered protein. J. Mol. Graph. Model. 19, 26–59. [17] Uversky, V.N. and Dunker, A.K. (2010) Understanding protein non-folding. Biochim. Biophys. Acta 1804, 1231–1264.

Please cite this article in press as: Uversky, V.N. The multifaceted roles of intrinsic disorder in protein complexes. FEBS Lett. (2015), http://dx.doi.org/ 10.1016/j.febslet.2015.06.004

8

V.N. Uversky / FEBS Letters xxx (2015) xxx–xxx

[18] Dunker, A.K. et al. (2008) The unfoldomics decade: an update on intrinsically disordered proteins. BMC Genomics 9 (Suppl. 2), S1. [19] Dunker, A.K. and Uversky, V.N. (2008) Signal transduction via unstructured protein conduits. Nat. Chem. Biol. 4, 229–230. [20] Uversky, V.N. (2011) Multitude of binding modes attainable by intrinsically disordered proteins: a portrait gallery of disorder-based complexes. Chem. Soc. Rev. 40, 1623–1634. [21] Uversky, V.N. (2013) Intrinsic disorder-based protein interactions and their modulators. Curr. Pharm. Des. 19, 4191–4213. [22] Patil, A. and Nakamura, H. (2006) Disordered domains and high surface charge confer hubs with the ability to interact with multiple proteins in interaction networks. FEBS Lett. 580, 2041–2045. [23] Haynes, C. et al. (2006) Intrinsic disorder is a common feature of hub proteins from four eukaryotic interactomes. PLoS Comput. Biol. 2, e100. [24] Ekman, D., Light, S., Bjorklund, A.K. and Elofsson, A. (2006) What properties characterize the hub proteins of the protein–protein interaction network of Saccharomyces cerevisiae? Genome Biol. 7, R45. [25] Dosztanyi, Z., Chen, J., Dunker, A.K., Simon, I. and Tompa, P. (2006) Disorder and sequence repeats in hub proteins and their implications for network evolution. J. Proteome Res. 5, 2985–2995. [26] Singh, G.P., Ganapathi, M., Sandhu, K.S. and Dash, D. (2006) Intrinsic unstructuredness and abundance of PEST motifs in eukaryotic proteomes. Proteins 62, 309–315. [27] Uversky, V.N., Gillespie, J.R. and Fink, A.L. (2000) Why are ‘‘natively unfolded’’ proteins unstructured under physiologic conditions? Proteins 41, 415–427. [28] Uversky, V.N. (2013) Unusual biophysics of intrinsically disordered proteins. Biochim. Biophys. Acta 1834, 932–951. [29] Dunker, A.K. and Obradovic, Z. (2001) The protein trinity-linking function and disorder. Nat. Biotechnol. 19, 805–806. [30] Uversky, V.N. (2002) Natively unfolded proteins: a point where biology waits for physics. Protein Sci. 11, 739–756. [31] Uversky, V.N. (2002) What does it mean to be natively unfolded? Eur. J. Biochem. 269, 2–12. [32] Hsu, W.L., Oldfield, C., Meng, J., Huang, F., Xue, B., Uversky, V.N., Romero, P. and Dunker, A.K. (2012) Intrinsic protein disorder and protein–protein interactions. Pac. Symp. Biocomput. 2012, 116–127. [33] Tompa, P. (2005) The interplay between structure and function in intrinsically unstructured proteins. FEBS Lett. 579, 3346–3354. [34] Teschke, C.M. and King, J. (1992) Folding and assembly of oligomeric proteins in Escherichia coli. Curr. Opin. Biotechnol. 3, 468–473. [35] Xu, D., Tsai, C.J. and Nussinov, R. (1998) Mechanism and evolution of protein dimerization. Protein Sci. 7, 533–544. [36] Gunasekaran, K., Tsai, C.J. and Nussinov, R. (2004) Analysis of ordered and disordered protein complexes reveals structural features discriminating between stable and unstable monomers. J. Mol. Biol. 341, 1327–1341. [37] Fuxreiter, M., Toth-Petroczy, A., Kraut, D.A., Matouschek, A.T., Lim, R.Y., Xue, B., Kurgan, L. and Uversky, V.N. (2014) Disordered proteinaceous machines. Chem. Rev. 114, 6806–6843. [38] Peng, Z., Oldfield, C.J., Xue, B., Mizianty, M.J., Dunker, A.K., Kurgan, L. and Uversky, V.N. (2014) A creature with a hundred waggly tails: intrinsically disordered proteins in the ribosome. Cell. Mol. Life Sci. 71, 1477–1504. [39] Peng, Z., Mizianty, M.J., Xue, B., Kurgan, L. and Uversky, V.N. (2012) More than just tails: intrinsic disorder in histone proteins. Mol. Biosyst. 8, 1886–1901. [40] Kussie, P.H., Gorina, S., Marechal, V., Elenbaas, B., Moreau, J., Levine, A.J. and Pavletich, N.P. (1996) Structure of the MDM2 oncoprotein bound to the p53 tumor suppressor transactivation domain. Science 274, 948–953. [41] Uesugi, M., Nyanguile, O., Lu, H., Levine, A.J. and Verdine, G.L. (1997) Induced alpha helix in the VP16 activation domain upon binding to a human TAF. Science 277, 1310–1313. [42] Radhakrishnan, I., PerezAlvarado, G.C., Parker, D., Dyson, H.J., Montminy, M.R. and Wright, P.E. (1997) Solution structure of the KIX domain of CBP bound to the transactivation domain of CREB: a model for activator: coactivator interactions. Cell 91, 741–752. [43] Sugase, K., Dyson, H.J. and Wright, P.E. (2007) Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447, 1021. U11. [44] Papoian, G.A. and Wolynes, P.G. (2003) The physics and bioinformatics of binding and folding – an energy landscape perspective. Biopolymers 68, 333– 349. [45] Shirai, N.C. and Kikuchi, M. (2013) Structural flexibility of intrinsically disordered proteins induces stepwise target recognition. J. Phys. Chem. 139, 225103. [46] Zhang, Q., Yu, D., Seo, S., Stone, E.M. and Sheffield, V.C. (2012) Intrinsic protein–protein interaction-mediated and chaperonin-assisted sequential assembly of stable Bardet–Biedl syndrome protein complex, the BBSome. J. Biol. Chem. 287, 20625–20635. [47] Lucker, B.F., Behal, R.H., Qin, H., Siron, L.C., Taggart, W.D., Rosenbaum, J.L. and Cole, D.G. (2005) Characterization of the intraflagellar transport complex B core: direct interaction of the IFT81 and IFT74/72 subunits. J. Biol. Chem. 280, 27688–27696. [48] Hirano, Y. et al. (2008) Dissecting beta-ring assembly pathway of the mammalian 20S proteasome. EMBO J. 27, 2204–2213. [49] Sahasranaman, A., Dembowski, J., Strahler, J., Andrews, P., Maddock, J. and Woolford Jr., J.L. (2011) Assembly of Saccharomyces cerevisiae 60S ribosomal subunits: role of factors required for 27S pre-rRNA processing. EMBO J. 30, 4020–4032.

[50] Marsh, J.A., Hernandez, H., Hall, Z., Ahnert, S.E., Perica, T., Robinson, C.V. and Teichmann, S.A. (2013) Protein complexes are under evolutionary selection to assemble via ordered pathways. Cell 153, 461–470. [51] Marsh, J.A. and Teichmann, S.A. (2013) Parallel dynamics and evolution: protein conformational fluctuations and assembly reflect evolutionary changes in sequence and structure. BioEssays 36, 209–218. [52] Hall, Z., Hernandez, H., Marsh, J.A., Teichmann, S.A. and Robinson, C.V. (2013) The role of salt bridges, charge density, and subunit flexibility in determining disassembly routes of protein complexes. Structure 21, 1325–1337. [53] Hernandez, H. and Robinson, C.V. (2007) Determining the stoichiometry and interactions of macromolecular assemblies from mass spectrometry. Nat. Protoc. 2, 715–726. [54] Levy, E.D., Boeri Erba, E., Robinson, C.V. and Teichmann, S.A. (2008) Assembly reflects evolution of protein complexes. Nature 453, 1262–1265. [55] Hurley, T.D., Yang, J., Zhang, L., Goodwin, K.D., Zou, Q., Cortese, M., Dunker, A.K. and DePaoli-Roach, A.A. (2007) Structural basis for regulation of protein phosphatase 1 by inhibitor-2. J. Biol. Chem. 282, 28874–28883. [56] Kiss, R. et al. (2008) Calcium-induced tripartite binding of intrinsically disordered calpastatin to its cognate enzyme, calpain. FEBS Lett. 582, 2149– 2154. [57] Galea, C.A., Nourse, A., Wang, Y., Sivakolundu, S.G., Heller, W.T. and Kriwacki, R.W. (2008) Role of intrinsic flexibility in signal transduction mediated by the cell cycle regulator, p27 Kip1. J. Mol. Biol. 376, 827–838. [58] Galea, C.A., Wang, Y., Sivakolundu, S.G. and Kriwacki, R.W. (2008) Regulation of cell division by intrinsically unstructured proteins: intrinsic flexibility, modularity, and signaling conduits. Biochemistry 47, 7598–7609. [59] Graham, T.A., Weaver, C., Mao, F., Kimelman, D. and Xu, W. (2000) Crystal structure of a beta-catenin/Tcf complex. Cell 103, 885–896. [60] Luscombe, N.M., Austin, S.E., Berman, H.M. and Thornton, J.M. (2000) An overview of the structures of protein–DNA complexes. Genome Biol. 1. REVIEWS001. [61] Reeves, R. (2001) Molecular biology of HMGA proteins: hubs of nuclear function. Gene 277, 63–81. [62] Huth, J.R., Bewley, C.A., Nissen, M.S., Evans, J.N., Reeves, R., Gronenborn, A.M. and Clore, G.M. (1997) The solution structure of an HMG-I(Y)-DNA complex defines a new architectural minor groove binding motif. Nat. Struct. Biol. 4, 657–665. [63] Reeves, R. and Nissen, M.S. (1990) The A.T-DNA-binding domain of mammalian high mobility group I chromosomal proteins. A novel peptide motif for recognizing DNA structure. J. Biol. Chem. 265, 8573–8582. [64] Carter, A.P., Clemons, W.M., Brodersen, D.E., Morgan-Warren, R.J., Wimberly, B.T. and Ramakrishnan, V. (2000) Functional insights from the structure of the 30S ribosomal subunit and its interactions with antibiotics. Nature 407, 340–348. [65] Wimberly, B.T., Brodersen, D.E., Clemons Jr., W.M., Morgan-Warren, R.J., Carter, A.P., Vonrhein, C., Hartsch, T. and Ramakrishnan, V. (2000) Structure of the 30S ribosomal subunit. Nature 407, 327–339. [66] Brodersen, D.E., Clemons Jr., W.M., Carter, A.P., Wimberly, B.T. and Ramakrishnan, V. (2002) Crystal structure of the 30 S ribosomal subunit from Thermus thermophilus: structure of the proteins and their interactions with 16 S RNA. J. Mol. Biol. 316, 725–768. [67] Lukhele, S., Bah, A., Lin, H., Sonenberg, N. and Forman-Kay, J.D. (2013) Interaction of the eukaryotic initiation factor 4E with 4E-BP2 at a dynamic bipartite interface. Structure 21, 2186–2196. [68] Mader, S., Lee, H., Pause, A. and Sonenberg, N. (1995) The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 gamma and the translational repressors 4E-binding proteins. Mol. Cell. Biol. 15, 4990–4997. [69] Fletcher, C.M., McGuire, A.M., Gingras, A.C., Li, H., Matsuo, H., Sonenberg, N. and Wagner, G. (1998) 4E binding proteins inhibit the translation factor eIF4E without folded structure. Biochemistry 37, 9–15. [70] Abiko, F., Tomoo, K., Mizuno, A., Morino, S., Imataka, H. and Ishida, T. (2007) Binding preference of eIF4E for 4E-binding protein isoform and function of eIF4E N-terminal flexible region for interaction, studied by SPR analysis. Biochem. Biophys. Res. Commun. 355, 667–672. [71] Fuxreiter, M. and Tompa, P. (2012) Fuzzy complexes: a more stochastic view of protein function. Adv. Exp. Med. Biol. 725, 1–14. [72] R. Hendus-Altenburger, E. Pedraz-Cuesta, J.A. Schnell, E. Papaleo, C.W. Olesen, J.M. la Cour, S.F. Pedersen, B.B. Kragelund, Scaffolding by docking domains link disorder in human Na+/H+ Exchanger 1 to regulation of Extracellular Signal Regulated Kinase 2 (2015). [73] Perham, R.N. (1975) Self-assembly of biological macromolecules. Philos. Trans. R. Soc. Lond. Ser. B Biol. Sci. 272, 123–136. [74] Barbar, E. and Nyarko, A. (2015) Polybivalency and disordered proteins in ordering macromolecular assemblies. Semin. Cell Dev. Biol. 37, 20–25. [75] Mourez, M., Kane, R.S., Mogridge, J., Metallo, S., Deschatelets, P., Sellman, B.R., Whitesides, G.M. and Collier, R.J. (2001) Designing a polyvalent inhibitor of anthrax toxin. Nat. Biotechnol. 19, 958–961. [76] Mourez, M. and Collier, R.J. (2004) Use of phage display and polyvalency to design inhibitors of protein–protein interactions. Methods Mol. Biol. 261, 213–228. [77] Leppla, S.H. (1995) Anthrax toxins. In Bacterial toxins and virulence factors in diseases (Moss, J., Iglewski, B., Vaughan, M. and Tu, A., Eds.), Handbook of Natural Toxins, pp. 543–572, Dekker, New York, NY. [78] Duesbery, N.S. et al. (1998) Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science 280, 734–737.

Please cite this article in press as: Uversky, V.N. The multifaceted roles of intrinsic disorder in protein complexes. FEBS Lett. (2015), http://dx.doi.org/ 10.1016/j.febslet.2015.06.004

V.N. Uversky / FEBS Letters xxx (2015) xxx–xxx [79] Dunker, A.K., Cortese, M.S., Romero, P., Iakoucheva, L.M. and Uversky, V.N. (2005) Flexible nets: the roles of intrinsic disorder in protein interaction networks. FEBS J. 272, 5129–5148. [80] Tompa, P. (2010) Structure and Function of Intrinsically Disordered Proteins, CRC Press, Boca Raton. [81] Buday, L. and Tompa, P. (2010) Functional classification of scaffold proteins and related molecules. FEBS J. 277, 4348–4355. [82] Cortese, M.S., Uversky, V.N. and Dunker, A.K. (2008) Intrinsic disorder in scaffold proteins: getting more from less. Prog. Biophys. Mol. Biol. 98, 85– 106. [83] Balazs, A. et al. (2009) High levels of structural disorder in scaffold proteins as exemplified by a novel neuronal protein, CASK-interactive protein1. FEBS J. 276, 3744–3756. [84] Kriwacki, R.W., Hengst, L., Tennant, L., Reed, S.I. and Wright, P.E. (1996) Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity. Proc. Natl. Acad. Sci. U.S.A. 93, 11504–11509. [85] Lacy, E.R., Filippov, I., Lewis, W.S., Otieno, S., Xiao, L., Weiss, S., Hengst, L. and Kriwacki, R.W. (2004) P27 binds cyclin-CDK complexes through a sequential mechanism involving binding-induced protein folding. Nat. Struct. Mol. Biol. 11, 358–364. [86] Lacy, E.R. et al. (2005) Molecular basis for the specificity of p27 toward cyclin-dependent kinases that regulate cell division. J. Mol. Biol. 349, 764– 773. [87] Oldfield, C.J., Cheng, Y., Cortese, M.S., Romero, P., Uversky, V.N. and Dunker, A.K. (2005) Coupled folding and binding with alpha-helix-forming molecular recognition elements. Biochemistry 44, 12454–12470. [88] Cheng, Y., Oldfield, C.J., Meng, J., Romero, P., Uversky, V.N. and Dunker, A.K. (2007) Mining alpha-helix-forming molecular recognition features with cross species sequence alignments. Biochemistry 46, 13468–13477. [89] Mohan, A. (2006) MoRFs: a dataset of molecular recognition featuresThe School of Informatics, Indiana University, Indianapolis. pp. 59. [90] Vacic, V., Oldfield, C.J., Mohan, A., Radivojac, P., Cortese, M.S., Uversky, V.N. and Dunker, A.K. (2007) Characterization of molecular recognition features, MoRFs, and their binding partners. J. Proteome Res. 6, 2351–2366. [91] Uversky, V.N. and Dunker, A.K. (2010) Understanding protein non-folding. Biochim. Biophys. Acta 1804, 1231–1264. [92] Tompa, P. and Fuxreiter, M. (2008) Fuzzy complexes: polymorphism and structural disorder in protein–protein interactions. Trends Biochem. Sci. 33, 2–8. [93] Hazy, E. and Tompa, P. (2009) Limitations of induced folding in molecular recognition by intrinsically disordered proteins. ChemPhysChem 10, 1415– 1419. [94] Sigalov, A., Aivazian, D. and Stern, L. (2004) Homooligomerization of the cytoplasmic domain of the T cell receptor zeta chain and of other proteins containing the immunoreceptor tyrosine-based activation motif. Biochemistry 43, 2049–2061. [95] Sigalov, A.B., Zhuravleva, A.V. and Orekhov, V.Y. (2007) Binding of intrinsically disordered proteins is not necessarily accompanied by a structural transition to a folded form. Biochimie 89, 419–421. [96] Permyakov, S.E., Millett, I.S., Doniach, S., Permyakov, E.A. and Uversky, V.N. (2003) Natively unfolded C-terminal domain of caldesmon remains substantially unstructured after the effective binding to calmodulin. Proteins 53, 855–862. [97] Jakob, U., Kriwacki, R. and Uversky, V.N. (2014) Conditionally and transiently disordered proteins: awakening cryptic disorder to regulate protein function. Chem. Rev. 114, 6779–6805. [98] Bardwell, J.C. and Jakob, U. (2012) Conditional disorder in chaperone action. Trends Biochem. Sci. 37, 517–525. [99] Creamer, T.P. (2013) Transient disorder. Calcineurin as an example. Intrinsically Disordered. Proteins 1, e26412. [100] Xue, B. et al. (2013) Stochastic machines as a colocalization mechanism for scaffold protein function. FEBS Lett. 587, 1587–1591. [101] Zhang, Y., Qiu, W.J., Chan, S.C., Han, J., He, X. and Lin, S.C. (2002) Casein kinase I and casein kinase II differentially regulate axin function in Wnt and JNK pathways. J. Biol. Chem. 277, 17706–17712. [102] Dajani, R., Fraser, E., Roe, S.M., Yeo, M., Good, V.M., Thompson, V., Dale, T.C. and Pearl, L.H. (2003) Structural basis for recruitment of glycogen synthase kinase 3beta to the axin-APC scaffold complex. EMBO J. 22, 494–501. [103] Xing, Y., Takemaru, K., Liu, J., Berndt, J.D., Zheng, J.J., Moon, R.T. and Xu, W. (2008) Crystal structure of a full-length beta-catenin. Structure 16, 478–487. [104] Borg, M., Mittag, T., Pawson, T., Tyers, M., Forman-Kay, J.D. and Chan, H.S. (2007) Polyelectrostatic interactions of disordered ligands suggest a physical basis for ultrasensitivity. Proc. Natl. Acad. Sci. U.S.A. 104, 9650–9655. [105] Mittag, T. et al. (2008) Dynamic equilibrium engagement of a polyvalent ligand with a single-site receptor. Proc. Natl. Acad. Sci. U.S.A. 105, 17772– 17777. [106] Mittag, T., Kay, L.E. and Forman-Kay, J.D. (2010) Protein dynamics and conformational disorder in molecular recognition. J. Mol. Recogn. 23, 105– 116. [107] Mittag, T., Marsh, J., Grishaev, A., Orlicky, S., Lin, H., Sicheri, F., Tyers, M. and Forman-Kay, J.D. (2010) Structure/function implications in a dynamic complex of the intrinsically disordered Sic1 with the Cdc4 subunit of an SCF ubiquitin ligase. Structure 18, 494–506.

9

[108] Nash, P. et al. (2001) Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature 414, 514–521. [109] Uversky, V.N., Kuznetsova, I.M., Turoverov, K.K. and Zaslavsky, B. (2015) Intrinsically disordered proteins as crucial constituents of cellular aqueous two phase systems and coacervates. FEBS Lett. 589, 15–22. [110] Phair, R.D. and Misteli, T. (2000) High mobility of proteins in the mammalian cell nucleus. Nature 404, 604–609. [111] Pederson, T. (2001) Protein mobility within the nucleus – what are the right moves? Cell 104, 635–638. [112] Maul, G.G., Negorev, D., Bell, P. and Ishov, A.M. (2000) Review: properties and assembly mechanisms of ND10, PML bodies, or PODs. J. Struct. Biol. 129, 278–287. [113] Huang, S. (2000) Review: perinucleolar structures. J. Struct. Biol. 129, 233– 240. [114] Fox, A.H., Lam, Y.W., Leung, A.K., Lyon, C.E., Andersen, J., Mann, M. and Lamond, A.I. (2002) Paraspeckles: a novel nuclear domain. Curr. Biol. 12, 13– 25. [115] Lamond, A.I. and Spector, D.L. (2003) Nuclear speckles: a model for nuclear organelles. Nat. Rev. Mol. Cell Biol. 4, 605–612. [116] Shav-Tal, Y., Blechman, J., Darzacq, X., Montagna, C., Dye, B.T., Patton, J.G., Singer, R.H. and Zipori, D. (2005) Dynamic sorting of nuclear components into distinct nucleolar caps during transcriptional inhibition. Mol. Biol. Cell 16, 2395–2413. [117] Decker, C.J., Teixeira, D. and Parker, R. (2007) Edc3p and a glutamine/ asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae. J. Cell Biol. 179, 437–449. [118] Brangwynne, C.P., Eckmann, C.R., Courson, D.S., Rybarska, A., Hoege, C., Gharakhani, J., Julicher, F. and Hyman, A.A. (2009) Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729–1732. [119] Chuma, S., Hosokawa, M., Tanaka, T. and Nakatsuji, N. (2009) Ultrastructural characterization of spermatogenesis and its evolutionary conservation in the germline: germinal granules in mammals. Mol. Cell. Endocrinol. 306, 17–23. [120] Strzelecka, M., Trowitzsch, S., Weber, G., Luhrmann, R., Oates, A.C. and Neugebauer, K.M. (2010) Coilin-dependent snRNP assembly is essential for zebrafish embryogenesis. Nat. Struct. Mol. Biol. 17, 403–409. [121] Decker, M., Jaensch, S., Pozniakovsky, A., Zinke, A., O’Connell, K.F., Zachariae, W., Myers, E. and Hyman, A.A. (2011) Limiting amounts of centrosome material set centrosome size in C. elegans embryos. Curr. Biol. 21, 1259–1267. [122] Wippich, F., Bodenmiller, B., Trajkovska, M.G., Wanka, S., Aebersold, R. and Pelkmans, L. (2013) Dual specificity kinase DYRK3 couples stress granule condensation/dissolution to mTORC1 signaling. Cell 152, 791–805. [123] Brangwynne, C.P. (2013) Phase transitions and size scaling of membrane-less organelles. J. Cell Biol. 203, 875–881. [124] Keating, C.D. (2012) Aqueous phase separation as a possible route to compartmentalization of biological molecules. Acc. Chem. Res. 45, 2114– 2124. [125] Nott, T.J. et al. (2015) Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol. Cell 57, 936–947. [126] Weber, T., Zemelman, B.V., McNew, J.A., Westermann, B., Gmachl, M., Parlati, F., Sollner, T.H. and Rothman, J.E. (1998) SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772. [127] Sudhof, T.C. and Rothman, J.E. (2009) Membrane fusion: grappling with SNARE and SM proteins. Science 323, 474–477. [128] Weninger, K., Bowen, M.E., Chu, S. and Brunger, A.T. (2003) Single-molecule studies of SNARE complex assembly reveal parallel and antiparallel configurations. Proc. Natl. Acad. Sci. U.S.A. 100, 14800–14805. [129] Sollner, T., Whiteheart, S.W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P. and Rothman, J.E. (1993) SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318–324. [130] Hanson, P.I., Roth, R., Morisaki, H., Jahn, R. and Heuser, J.E. (1997) Structure and conformational changes in NSF and its membrane receptor complexes visualized by quick-freeze/deep-etch electron microscopy. Cell 90, 523–535. [131] Sutton, R.B., Fasshauer, D., Jahn, R. and Brunger, A.T. (1998) Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution. Nature 395, 347–353. [132] Zhang, X., Ma, L. and Zhang, Y. (2013) High-resolution optical tweezers for single-molecule manipulation. Yale J. Biol. Med. 86, 367–383. [133] Dutertre, M., Lambert, S., Carreira, A., Amor-Gueret, M. and Vagner, S. (2014) DNA damage: RNA-binding proteins protect from near and far. Trends Biochem. Sci. 39, 141–149. [134] Thalhammer, A., Bryant, G., Sulpice, R. and Hincha, D.K. (2014) Disordered cold regulated15 proteins protect chloroplast membranes during freezing through binding and folding, but do not stabilize chloroplast enzymes in vivo. Plant Physiol. 166, 190–201. [135] Hartmann-Petersen, R., Hendil, K.B. and Gordon, C. (2003) Ubiquitin binding proteins protect ubiquitin conjugates from disassembly. FEBS Lett. 535, 77– 81. [136] Ben-Shem, A., Garreau de Loubresse, N., Melnikov, S., Jenner, L., Yusupova, G. and Yusupov, M. (2011) The structure of the eukaryotic ribosome at 3.0 A resolution. Science 334, 1524–1529. [137] Shibata, N. et al. (2007) Crystallization and preliminary X-ray crystallographic studies of the axin DIX domain. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 63, 529–531.

Please cite this article in press as: Uversky, V.N. The multifaceted roles of intrinsic disorder in protein complexes. FEBS Lett. (2015), http://dx.doi.org/ 10.1016/j.febslet.2015.06.004