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Modulation of IntrinsicallyDisordered Protein Function byPost-translational ModificationsPublished, JBC Papers in Press, February 5, 2016, DOI 10.1074/jbc.R115.695056

Alaji Bah‡§1 and Julie D. Forman-Kay‡§2

From the ‡Program in Molecular Structure & Function, The Hospital for SickChildren, Toronto, Ontario M5G 0A4 and the §Department of Biochemistry,University of Toronto, Toronto, Ontario M5S 1A8, Canada

Post-translational modifications (PTMs) produce significantchanges in the structural properties of intrinsically disorderedproteins (IDPs) by affecting their energy landscapes. PTMs caninduce a range of effects, from local stabilization or destabi-lization of transient secondary structure to global disorder-to-order transitions, potentially driving complete statechanges between intrinsically disordered and folded states ordispersed monomeric and phase-separated states. Here, wediscuss diverse biological processes that are dependent onPTM regulation of IDPs. We also present recent tools forgenerating homogenously modified IDPs for studies of PTM-mediated IDP regulatory mechanisms.

The amino acid sequences of intrinsically disordered pro-teins or protein regions (often simply referred to as IDPs)3

determine their inability to fold into stable tertiary structuresunder physiological conditions and instead enable them to rap-idly interconvert between distinct conformations to mediatecritical biological functions (1, 2). The amino acid compositionsof IDPs range from very low complexity with little diversity inresidue types to much higher sequence complexity that enablesdisorder-to-order transitions upon binding or post-transla-tional modifications (PTMs) (3–5). This range of sequencecomposition leads to heterogeneous ensembles with variablehydrodynamic properties and fluctuating secondary and terti-ary structure, with some IDPs able to self-associate in phase-separated protein-dense droplets (6 –11). Structural heteroge-neity and dynamic fluctuations endow IDPs with uniqueadvantages over folded proteins for certain roles (12–14). IDPsexpose, at least transiently, their entire primary sequence forbinding, enabling multiple interactions along their polypeptidechains. This makes them hubs in protein complexes and inte-

grators in signaling networks (15–19). IDPs also facilitate themultivalent interactions that drive phase separation, underly-ing cellular membraneless organelles and signaling puncta(20 –22).

Due to their accessibility to modifying enzymes, IDPs are thepredominant sites of PTMs, which significantly expands theirfunctional versatility (3, 23). By changing the physicochemicalproperties of the primary sequence, PTMs induce a range ofstructural changes, from local stabilization or destabilization oftransient secondary structure to more global conformationalchanges in disorder-to-order transitions (24, 25). As the hydro-dynamic properties of IDPs are strongly affected by electro-static effects, PTMs that change charge (e.g. phosphorylationand acetylation) can modulate compactness (9 –11). PTMs canalso lead to complete state changes, between disordered andfolded states (26 –28) or dispersed monomeric and phase-sep-arated states (6, 8, 22). The scope of PTM-mediated structuraland dynamic changes described in recent biophysical and bio-chemical studies of IDPs is similar to those due to binding toother proteins, nucleic acids, lipids, carbohydrates, ions, cofac-tors, and other small molecules (4, 29, 30).

Here we review the effects of PTMs on IDP structure andfunction and how such effects regulate fundamental biologicalprocesses, from PTM-mediated conformational transitions ofindividual IDPs to the assembly of multi-protein complexes andthe formation of membraneless protein organelles. We begin bypresenting the diversity of PTMs and their effects on IDP struc-ture and function and then focus on phosphorylation, one ofthe most common PTMs. Because the investigation of PTMeffects on IDPs is often limited by the ability to generate homo-geneously modified samples, we discuss recent advances inrecombinant protein expression systems and chemical synthe-sis strategies for producing modified IDP samples. Theseenable detailed biochemical and biophysical characterizationusing general methods for IDPs as well as those specific for IDPswith PTMs (4, 30, 31)

Diversifying the Structural Properties of IDPs via PTMs

Biological systems evolved multiple types of PTMs to expandthe functional diversity of proteins, particularly of IDPs (3, 32,33). Usually, PTM involves the addition of chemical functionalgroups, including phosphoryl, alkyl, glycosyl, and acyl groups,or small proteins such as ubiquitin and SUMO. In other cases,PTM involves modifying the chemical properties of aminoacids through oxidation, deimidation, and deamidation. Thesemodifications generate proteins with “new” amino acid compo-sitions because each PTM results in novel chemical and stericproperties that do not exist in the other standard set of 20amino acids. In contrast, PTMs such as cis-trans isomerizationof prolines, epimerization of serines, intein-based protein splic-ing, and the cleavage of the polypeptide chain at specific sites byproteases do not change the chemical nature of the amino acidside chains. IDPs can have single or multiple PTMs or a com-bination of different PTMs on the same or different aminoacids, providing many layers of complex biological regulation

* This work was supported by grants from the Canadian Institutes of HealthResearch (CIHR), Canadian Cancer Society, and Cystic Fibrosis Canada (toJ. D. F. K.). This is the third article in the Thematic Minireview series “Intrin-sically Disordered Proteins.” The authors declare that they have no con-flicts of interest with the contents of this article.

1 Partially supported by a Restracomp award from the Hospital for Sick Chil-dren and a post-doctoral fellowship from CIHR. To whom correspondencemay be addressed. E-mail: [email protected].

2 To whom correspondence may be addressed. E-mail: [email protected] The abbreviations used are: IDP, intrinsically disordered protein; PTM, post-

translational modification; IDR, intrinsically disordered region; SUMO,small ubiquitin-like modifier; ETS, E26 transformation-specific; SRR, serine-rich region; IM, autoinhibitory module; 4E-BP, 4E-binding protein; UD,Unique domain; N-WASP, neuronal Wiskott–Aldrich Syndrome protein;NCL, native chemical ligation; p, phospho.

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 13, pp. 6696 –6705, March 25, 2016© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

6696 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 13 • MARCH 25, 2016

MINIREVIEW

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due to the additive, cooperative, or competitive responsesbetween these multiple modifications (34 –37). Although mostare catalyzed by specific enzymes, e.g. transferases and isomer-ases, some PTMs, including L-aspartate isomerization and L-as-paragine deamidation, occur spontaneously (38).

PTMs elicit diverse effects on the biological functions of IDPsby altering the energetics of their conformational landscapeand by modulating interactions with other cellular components(4, 31). PTMs can regulate IDP function by (i) altering theirsteric, hydrophobic, or electrostatic properties due to primarystructural effects (39); (ii) stabilizing, destabilizing, or eveninducing secondary structural elements (24); and (iii) inhibit-ing/enhancing long-range tertiary contacts between distalmotifs or PTM sites within the IDPs, or with interaction part-ners (40). PTMs can tune all aspects of IDPs, including thenature of the disordered ensemble (i.e. extended versus col-lapsed), the propensity (if any) of the secondary structural ele-ments, the types of transient intra-molecular tertiary contacts,the (in)ability to fold into stable three-dimensional structure orto interact with a binding partner or specific modular domainsthat interact with post-translationally modified residues duringsignal transduction, and the partitioning into membraneless,phase-separated, protein-dense cellular bodies (Figs. 1 and 2).Through these varied responses, biological signal inputs arewritten onto IDPs to change their functional output.

Phosphorylation: The Prototypical PTM

To provide insights into how PTMs elicit such effects on thestructure and ultimately function of IDPs, we focus on one ofthe most common PTMs, phosphorylation. At least a third ofeukaryotic proteins may be phosphorylated, and most phos-phorylation sites are within intrinsically disordered regions

FIGURE 1. Post-translational modifications of IDPs can induce diverse structural changes. PTM-mediated structural changes within IDP ensembles andbetween IDPs and folded states are shown. These conformational ensembles range from folded and molten globules to extended and collapsed disorderedensembles (represented with six members) with and without transient or stable secondary structural elements.

FIGURE 2. IDPs can form PTM-induced phase-separated droplets in cellu-lar organization such as membraneless organelles and signaling puncta.A and B, PTMs can enhance the assembly or disassembly of the phase-sepa-rated state (A) or control whether another molecule can be encapsulated intoor released from the droplet (B). C, weak multivalent interactions betweenIDPs with multiple PTM sites and proteins containing multiple PTM bindingmodules can also result in phase separation to form cellular bodies or signal-ing puncta.

MINIREVIEW: Modulation of IDP Function by PTMs

MARCH 25, 2016 • VOLUME 291 • NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 6697


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(IDRs) (41– 43). Phosphorylation and dephosphorylation ofIDPs by kinases and phosphatases, respectively, provide a majorregulatory mechanism in key eukaryotic processes, e.g. controlof cell cycle, transcription, splicing, and translation. About 2%of the human genome is dedicated to coding for over 500 pro-tein kinases, representing the largest class of PTM enzymes. Inmammals, �90% of phosphorylation occurs on serine with�10% on threonine or tyrosine.

Phosphorylation replaces a neutral hydroxyl (OH) groupwith a tetrahedral phosphoryl group (PO4

2�) bearing two nega-tive charges, drastically altering the steric, chemical, and elec-trostatic properties and introducing new interaction capabili-ties. Under physiological conditions, the double negativecharge of the phosphoryl moiety together with its large hydra-tion shell is chemically different from the acidic amino acids(Asp and Glu) often used as phospho-mimics (26, 44) that pos-sess small hydration shells and only a single negative charge.Phosphorylation provides novel possibilities for intra- andintermolecular electrostatic interactions, including salt bridges,via charge or hydrogen-bonding networks involving up to fourphosphoryl oxygens. Phosphorylated residues can form strongsalt bridges with the guanido moiety of arginines, which arebetter able to interact with phosphoryl groups than protonatedprimary amines of Lys side chains due to their rigid planarstructure and ability to form multiple H-bonds (45). The inter-action strength depends on the proximity, orientation, and sur-rounding environments of the phosphoryl and guanido groups,allowing fine-tuning by the neighboring amino acid composi-tion within the IDP. Phosphorylated residues can also interactwith helix dipoles. Phosphoserine is the most stabilizing aminoacid for �-helices when located at the Ncap, N1, N2, and N3positions, whereas it is destabilizing in the interior or at the Cterminus of the helix (46).

Phosphorylation provides novel sites for binding to modularphosphorylation-interaction domains, including SH2 and PTBfor pTyr-containing IDPs and 14-3-3, BRCT, FF, WW, WD40,MH2, FHA, polo box, and IRF3 for pSer/pThr-containing IDPs(47). The evolution of multiple, distinct structural domains forinteracting with phosphorylated residues reflects the impor-tance and the complexity of phosphorylation-mediated pro-tein-protein interactions in biology. Phosphorylation involves arich interplay between protein machineries containing kinases(writers), phosphatases (erasers), and phosphorylation-inter-action domains (readers) (48) with many varied biological con-sequences. Other types of PTMs elicit similar effects, but viadifferent physico-chemical properties using different writers,erasers, and readers. For example, acetylated lysine and methy-lated arginine within IDPs can interact with bromo domain-and tudor domain-containing proteins, respectively (49),affecting nucleic acid binding and RNA pathways.

Regulation of IDP Function in Biology

PTMs of IDPs provide key regulatory mechanisms in diversebiological processes, from control of cell cycle, transcription,splicing, and translation to cellular signaling and cellular orga-nization. Below, we present examples of PTM modulation ofIDP function by controlling the conformational transitions ofindividual proteins, enabling the docking of multi-protein com-

plexes, directing subcellular localization, and forming distinctmembraneless protein bodies.

Disorder-to-folding Transitions

Although IDPs lack the ability to fold into stable three-di-mensional structures on their own under physiological condi-tions, many IDPs undergo some degree of folding upon ligandbinding (2, 4, 50, 51). Such disorder-to-order transitions rangefrom the stabilization or induction of secondary structural ele-ments such as �-helices to the complete folding of an entire IDPupon binding to a partner. Binding of some IDPs to differentphysiological protein targets can even induce different foldedstates (4, 52–55). These observations demonstrate that changesin the surrounding physico-chemical environment can deter-mine the ability of IDPs to fold and the nature of the fold itself.PTMs, by altering the physical and chemical properties of IDPs,can also provide a mechanism for partial or complete folding ofIDRs or of entire IDPs, and these transitions can be exploitedfor regulating biology (Fig. 1). The reverse is also true in thatPTM of folded proteins or stable secondary structural elementscan lead to their unfolding or destabilization (27, 28). Althoughthere are numerous examples of structural changes upon PTMs(56 – 60), here we describe two examples in which multi-sitephosphorylation leads to conformational switching.

The E26 transformation-specific (ETS) domain-containingprotein family is one of the largest groups of transcription fac-tors with different members having distinct mechanisms of reg-ulating DNA recognition by their ETS domains (61) (Fig. 3A).The ETS domain of Ets-1 is regulated by a flanking autoinhibi-tory module (IM) and an adjacent disordered serine-rich region(SRR). The IM contains four �-helices (HI-1, HI-2, H4, and H5)that pack against the ETS domain on the surface opposite to theDNA binding interface and, upon DNA binding, IM becomesmore flexible with the entire HI-1 helix undergoing an order-to-disorder transition (62– 65). Transient SRR interactionswith both the IM and the ETS domain repress DNA binding.However, multiple phosphorylation of up to five sites in theSRR, as a result of Ca2� signaling, further reduces DNA bindingby dampening the flexibility of the SRR, the ETS domain, andthe IM, resulting in an allosterically induced disorder-to-ordertransition for HI-1. Physico-chemical analysis of the SRRrevealed that phosphorylation-induced autoinhibition is medi-ated by interactions between aromatic residues and phospho-serines, highlighting a novel intramolecular regulatory mecha-nism involving PTMs of IDPs (66).

Another example in which PTM-mediated conformationalswitching plays a critical role is that of the phosphorylation-induced folding of 4E-BP2, which controls cap-dependentmRNA translation initiation (Fig. 3B) (26). 4E-binding proteins(4E-BPs) inhibit translation by binding to eukaryotic initiationfactor (eIF) 4E, which is part of the tripartite eIF4F complex(eIF4E, eIF4G, and eIF4A) that recruits the 40S ribosome tomRNA (67). 4E-BP binding to eIF4E prevents the assembly ofeIF4F by competing with eIF4G because they interact at anoverlapping eIF4E surface using the same YXXXXL� canonicalbinding motif (68). Previous studies support the formation of anextended, bipartite, fuzzy interaction with eIF4E, with a disor-der-to-helix transition for the canonical binding motif (69 –72).




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Upon hierarchical multi-site phosphorylation of Thr37/Thr46,followed by Thr70, Ser65, and Ser83, the affinity of 4E-BP2 foreIF4E is significantly weakened, enabling eIF4G interaction and

translation initiation (73). Phosphorylation of Thr37/Thr46 of4E-BP2 reduces the affinity for eIF4E by inducing a disorder-to-order transition, resulting in the folding of residues Pro18–

FIGURE 3. IDP-mediated disorder-to-order transitions to regulate protein complex formation. A, top, schematic representation of Ets-1, composed of anN-terminal protein interaction domain (PNT, gray), the transactivation domain (TAD, gray) followed by a C-terminal SRR (blue), and the IM domain (cyan)flanking the ETS DNA binding domain (red/yellow). Lower, in the apo state, helix HI-1 of the IM domain is in dynamic equilibrium between folded and disorderedconformations (Protein Data Bank (PDB) codes 1R36 (65) and 1MDM (64), respectively). Upon multi-site phosphorylation on the SRR (blue to red circles), SRRdynamic fluctuations are damped, enhancing transient stabilizing interactions with the ETS and IM domains, further reducing DNA binding. B, regulation of the4E-BP2�eIF4E complex by phosphorylation of Thr37 (pT37) and Thr46 (pT46) (orange stick model) leads to folding of residues Pro18–Arg62 (blue, PDB code: 2MX4),with further phosphorylation at Ser65, Thr70, and Ser83 stabilizing the folded state, enabling translation initiation. In the absence of eIF4E, non-phosphorylated4E-BP2 (black) is disordered albeit with significant transient secondary structural elements (69). 4E-BP2 utilizes residues from about Tyr34 to Asp90 (green)consisting of a helical element (red) containing the canonical binding (54YXXXXL�60) motif and a flexible secondary binding site involving 78IPGVT82 for eIF4E(brown surface) binding. (Note: The longest observed fragment of a 4E-BP in complex with eIF4E in a crystal structure (72) is only Met49 to Ser82 (PDB code:4UED).) This dynamic complex is represented by an ensemble of three conformers of 4E-BP2 on the surface of eIF4E.




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Arg62 of 4E-BP2 into a four-stranded �-domain that is incom-patible with binding, whereas phosphorylation of Ser65, Thr70,and Ser83 stabilizes that folded domain (26). The discovery ofthe phosphorylation-mediated folding of 4E-BPs exemplifiesanother mode of biological regulation mediated by IDP PTM.

PTMs of Histone Tails to Generate a Histone Code forChromatin Regulation

Another biological process modulated by PTMs of IDRs ischromatin regulation. The fundamental building block of chro-matin is the nucleosome core particle: �147 bases of DNAwrapped around an octamer of two copies each of H2A, H2B,H3, and H4 proteins. PTM of the N- and C-terminal IDRs ofhistones (histone tails) is a major mechanism for regulating theaccessibility of the DNA within chromatin (74). PTM of histonetails is a dynamic process that transduces input signals from thecellular environment to regulate diverse genomic functionsincluding DNA replication, transcription, repair, and recombi-nation. Histones undergo a diverse array of PTMs includingacetylation, methylation, ubiquitination, and sumoylation oflysines; methylation and citrullination of arginines; phosphor-ylation of serines, threonines, and tyrosines; and isomerizationof prolines. For instance, acetylation of histone lysine residuesneutralizes the positive charge on the lysine and produces pro-found changes in the interaction between histones and nega-tively charged DNA molecules (75). Acetylation and methyla-tion can also allow the docking of protein machinery such aschromatin remodeling complexes that contain bromo andtudor domain interaction modules (49). Recent advances inNMR spectroscopy enabled the atomic-level structural anddynamic characterization of the nucleosome core particle andits complex formation (76), paving the way for future charac-terization of these diverse PTMs on the nucleosome. Futureresearch will need to elucidate crosstalk between these differenthistone PTMs with respect to chromatin structure andfunction.

Protein-Phospholipid Interactions

Many non-membrane proteins are tethered to phospholipidmembranes via PTM-mediated hydrophobic anchors such asmyristate, palmitate, isoprenoid, or glycosylphosphatidylinosi-tol groups (32). These PTMs enable subcellular localizationrequired for specific spatiotemporally restricted function(s).Another mechanism of protein-phospholipid interactioninvolves direct lipid binding by proteins in a process that is alsoregulated by PTMs. A potential synergy between these twomodes of protein-lipid interaction was recently demonstratedfor the non-receptor tyrosine kinase c-Src (Fig. 4A) (77). Srcfamily kinases are modular proteins consisting of a C-terminalcatalytic (Src homology 1 or SH1) domain, SH2 and SH3 inter-action domains, and two N-terminal IDRs, a Unique domain(UD) and an SH4 domain (78). c-Src either resides in the cyto-plasm or is attached to phospholipid membranes via a myristategroup attached to the SH4 Gly2, a PTM necessary but not suf-ficient for membrane anchoring. The SH4 using residues14RRR16 and the UD using the unique lipid binding region con-sisting of residues 60 – 67 can each bind phospholipids directly.However, PKA phosphorylation of the SH4 Ser17 and cyclin-de-

pendent kinase phosphorylation of Ser37/Thr75 of the UD dis-rupt such interactions (77). Therefore, multiple types of PTMsof the N-terminal IDRs of c-Src mediate its anchoring to themembrane, controlling how far into the cytoplasm the C-ter-minal kinase domain can “reach.”

One of the most dramatic examples of the effect of PTMs onIDRs in a physiological process is that of �-carboxylation of theGla domains of vitamin K-dependent clotting factors (Fig. 4B)(79). The blood coagulation cascade is the hemostatic mecha-nism that prevents loss of blood following vascular injury byactivating platelets and depositing fibrin, resulting in theiradhesion and aggregation at the site of damage. Key to the suc-cess of this process is the sequential assembly of vitamin K-de-pendent protease�cofactor complexes on a membrane surfaceto activate another vitamin K-dependent protease (80). Eachvitamin K-dependent clotting factor (factor X, factor IX, factorVII, prothrombin, protein C, protein S) contains an N-terminalIDR called the Gla domain, so named because 9 –12 of its glu-tamic acid residues are converted to �-carboxyglutamate,which sensitizes the Gla domain to a Ca2�-induced disorder-to-folding transition (81). Such folding endows clotting factorswith membrane surface binding properties (82). Assembly ofthese enzyme�cofactor�pro-enzyme complexes on a membranesurface enhances the catalytic activity between 5 and 6 orders ofmagnitude, significantly speeding up the clotting process toachieve the appropriate physiological response (80).

Modulation of Phase Separation

The physical partitioning of eukaryotic cells into functionallydistinct bodies and organelles provides separate environmentsthat allow diverse cellular processes to occur simultaneously.Although membrane-bound organelles, e.g. the nucleus, Golgiapparatus, and the mitochondrion, have been studied in exquis-ite detail, the structural and dynamic properties of membrane-less proteinaceous bodies have only begun to be characterized(Fig. 2) (6, 22, 83). Proteinaceous organelles are diverse in num-ber, size, and morphology, and they are present both in thenucleus (nucleolus, nuclear speckles, Cajal bodies, histonelocus body, and promyelocytic leukemia (PML) bodies) and inthe cytosol (stress granules, P-bodies, neuronal granules, andnuage) (8, 83). Phase-separated proteinaceous organelles canrapidly assemble or disassemble in response to subtle changesin the cellular environment, and they function prominently invarious RNA processes from transcription to degradation (7, 8).The central transporting component of the nuclear pore com-plex is another protein-dense structure created by phase sepa-ration of nucleoporin IDRs (22). Signaling puncta are examplesof more transient cellular features dependent on phase separa-tion, in this case relying on multivalent interactions of modularbinding domains to binding motifs within IDRs (20, 21).

Data emerging from this burgeoning field demonstrate thatmultiple, transient, and weak interactions drive formation ofdynamic liquid droplets or hydrogels, relying on elementswithin low complexity sequences of IDRs or domain-ligandinteractions. PTMs in the IDRs of these proteins have beendemonstrated to be an important regulatory mechanism formodulating their phase separation properties. For example,arginine methylation can regulate the phase separation of




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Ddx4, a major component of nuage (84) that plays a critical rolein the Piwi-interacting RNA pathway by protecting spermato-cytes and spermatids from deleterious activities of transposableelements (85). Ddx4 consists of a central DEAD box RNA heli-case domain and N- and C-terminal IDRs. These Ddx4 IDRsdirect its spontaneous self-association into membraneless pro-teinaceous organelles in HeLa cells and the isolated 250-residue

N-terminal IDR phase-separates in vitro (84). Alternatingcharge blocks and an over-representation of FG/GF andRG/GR motifs within the positively charged blocks appear to bekey to organelle formation. Phase-separated Ddx4 N terminusexcluded double-stranded DNA, but enriched single-strandedDNA in the interior of the droplets, suggesting that the phase-separated state has a functional role in nucleic acid biochemis-

FIGURE 4. PTM-regulated docking of IDRs onto a phospholipid membrane. A, anchoring of c-Src via SH4 and UD (77). Top, schematic representation of themodular domains of c-Src showing the IDRs SH4 and UD (blue) as well as the SH3 and SH2 interaction domains and a C-terminal tyrosine kinase domain (gray).In addition to the myristoyl group (green) attached to Gly2 of the SH4 domain, the SH4 domain and UD can also bind phospholipids. Phosphorylation of the SH4at Ser17 by PKA and of the UD at Thr37/Ser75 by cyclin-dependent kinase (CDK) controls the SH4 and Unique lipid binding region (ULBR) localization to thephospholipid membrane. B, �-carboxylation of the Gla domain enables the calcium-mediated disorder-to-folded transition of the Gla domain. Top, schematicrepresentation of factor IX with the Gla domain (red) followed by two tandem EGF domains and a C-terminal serine protease domain (gray). The N-terminalGlu-rich IDR undergoes multisite �-carboxylation to form the Gla domain (Gla residues shown in stick representation, PDB code: 1CFH). Upon Ca2� binding, theentire Gla domain folds into a four-helix bundle (calcium ions in magenta spheres, PDB codes: 1J35, 1CFI), which can bind phospholipid membranes. The Gladomain is represented by an ensemble of three conformers each color-coded N- to C-terminal from blue to red, respectively, with the C-terminal helixsuperimposed.




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try (Fig. 2B). PTM by asymmetric arginine dimethylation onknown conserved sites significantly inhibits phase separationby lowering the phase transition temperature by 25 °C. These invitro data suggest that PTM is a cellular mechanism for regu-lating nuage assembly/disassembly in response to physiologicalinput signals.

The phase separation that drives formation of signalingpuncta based on multivalent modular binding domain-ligandinteractions is also highly dependent on PTMs (Fig. 2C). Asindicated above, modular binding domains recognize PTMswithin IDRs, and these interactions are now implicated in phaseseparation. The degree of tyrosine phosphorylation (pTyr) onthe disordered cytoplasmic tail of nephrin controls the phasetransition of the multivalent three-component nephrin�NCK�N-WASP system. There are three potential nephrin pTyr sitesthat can each interact with the single SH2 domain of NCK,whereas the three SH3 domains of NCK can interact with thesix proline-rich motifs of N-WASP (20). Phase separationinduced by multivalent interactions of signaling proteinsresults in clustering of regulatory or regulated factors to facili-tate a specific biological process. For instance, in the presenceof the Arp2/3 complex, the phase separation of nephrin�NCK�N-WASP assembles actin filaments (21). These examples high-light the key roles PTM of IDRs play in regulating protein-basedcellular organization (Fig. 2).

Generating Post-translationally Modified IDP Samplesfor Biochemical and Biophysical Characterization

There are many experimental tools and computationalapproaches to study IDPs that are applicable to those withPTMs as well, including some that are specific for these modi-fied IDPs (4, 31). NMR spectroscopy, small angle x-ray scatter-ing, fluorescence, circular dichroism, and other data can beacquired, and a number of computational algorithms can beused (30). However, a major impediment for rigorous quantita-tive biochemical and biophysical structural and dynamic char-acterization of PTM-mediated effects on IDP structure andfunction is generating the large quantities of homogeneous andsite-specifically modified samples that are typically required forsuch studies. To overcome these challenges requires a combi-nation of traditional biochemistry, advanced molecular biologyapproaches, as well as improvements in chemical synthesis andsemisynthetic strategies (86 – 89). Here we describe three strat-egies for generating modified IDPs.

Expressing Post-translationally Modified IDPs UsingRecombinant DNA Technology

Advances in biochemical and molecular biology approacheshave dramatically increased the use of recombinant DNA tech-nology to design, express, and purify IDPs that are post-trans-lationally modified from a range of hosts including Escherichiacoli, yeast, plants, insects, or mammalian cell lines. PTMs can beintroduced after mRNA translation using modifying enzymeseither produced in a co-expression system (PTM within thecell) or expressed separately (in vitro PTM). Due to the possiblegeneration of non-homogenous or nonspecific modificationsfrom such modifying enzymes, a second approach involvingamber codon suppression technologies allows direct introduc-

tion of the PTM during mRNA translation with an orthogonaltRNA/aminoacyl-tRNA synthetase pair that geneticallyencodes an unnatural amino acid already containing the PTMof interest (90 –92). A more complex approach is to use anunnatural amino acid that contains a selective chemical tag forlater incorporation of the PTM (93). Protein expression sys-tems provide a relatively cheap and robust mechanism for gen-erating large quantities of high fidelity samples for a variety ofpeptides and proteins with homogenous PTMs, including ena-bling isotopic labeling (15N/13C/2H) for sophisticated NMR ormass spectrometry studies. The development of completelyrecoded or re-engineered expression hosts and advances in invitro protein expression systems will undoubtedly facilitate ourability to generate site-specific, homogenously modified IDPsamples in the future (94 –96).

Chemical Synthesis of Modified IDPs

One of the advantages of the chemical synthesis approach isthe ability to generate IDPs with single or multiple (identical orotherwise) PTMs located anywhere along the polypeptidechain (86). This strategy involves the generation of shorter pep-tide fragments containing the PTM(s) of interest and theirassembly into full-length IDP using the native chemical ligation(NCL) technique first introduced by Kent and co-workers (97).NCL is a chemoselective method based on the transthioesteri-fication of two polypeptide fragments, one with an N-terminalcysteine and the other with a C-terminal thioester, and it allowstwo fully unprotected peptide fragments to be reacted underneutral, aqueous conditions (86, 87).

Semisynthesis: A Mélange of Chemical Synthesis and ProteinExpression

Semisynthesis approaches are the most powerful methodsfor obtaining modified IDPs, as they combine the advantages ofchemical synthesis and protein expression to generate peptidefragments for the assembly of full-length IDPs using either theNCL or the expressed protein ligation techniques (98, 99). Forexample, the N-terminal peptide fragment containing a C-ter-minal thioester can be chemically synthesized (NCL) orobtained via intein-based protein expression, whereas theC-terminal fragment with an N-terminal cysteine residue canalso be chemically synthesized or expressed fused to a cleavabletag such as SUMO. As discussed above, these fragments can bemodified with PTM(s) of interest using either chemical or bio-chemical strategies prior to NCL or expressed protein ligation(98).

Conclusion

The number and diversity of post-translationally modifiedIDPs are astounding; over 300 distinct PTMs occur on eukary-otic proteins, and the human proteome contains up to a millionmodified peptide motifs (32, 33, 100). PTMs play extensiveroles in modulating the conformational properties and func-tions of IDPs. However, the structural, binding, and large-scaleassociation effects of modified IDPs for most of these are cur-rently unknown. State-of-the-art techniques in mass spectrom-etry and for generating homogeneously modified IDPs willincrease the identification of PTMs of IDPs under various phys-




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iological or pathological conditions and illuminate mechanisticdetails underlying their functional effects (100). Unveiling thestructural and functional consequences of modified IDPsrequires a combination of diverse disciplines ranging frommolecular biology, traditional biochemistry, and syntheticchemistry to sophisticated biophysical approaches includingNMR. Elucidating these PTM effects will have enormous ben-efits for expanding our understanding of complex biologicalsystems.

Acknowledgments—We thank Veronika Csizmok, Jennifer Dawson,Patrick Farber, and Mickaël Krzeminski for useful discussions andAndrew Chong for critical reading of the manuscript.

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Alaji Bah and Julie D. Forman-KayModifications

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