The
functional
importance
of
structure
in
unstructured
protein
regions
Norman
E
Davey
1,2,3Aftertwodecadesofresearch,intrinsicallydisorderedregions
(IDRs)areestablishedasawidespreadphenomenon.The
growingunderstandingofthesignificantfunctionalroleofIDRs
haschallengedthestructure–functionparadigm,proving
irrefutablythatastablyfoldedstructureisnotastrict
requirementforfunction.Nonetheless,(un)structure–function
relationshipsremainatthecoreofIDR-mediatedinteractions.
AnIDRcanpopulateacontinuouslytransitioningcontinuumof
structuralconformationsfromfullydisorderedtostable
globularstates.Intheseensembles,onlysubsetsof
conformationsarebindingcompetent,withintramolecularIDR
contactsservingasimportantintermolecularbinding
determinants.Here,wereviewourcurrentunderstandingof
differenttypesofintramolecularIDRinteractions,theireffects
onIDRcomplexformationandtheirmodesofbiological
regulation.
Addresses
1ConwayInstituteofBiomolecular&BiomedicalResearch,University College Dublin, Belfield, Dublin 4, Ireland
2Division of Cancer Biology, The Institute of Cancer Research, 237FulhamRoad,LondonSW36JB,UK
Correspondingauthor:Davey,NormanE([email protected]) 3Present address: Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK.
CurrentOpinioninStructuralBiology2019,56:155–163
ThisreviewcomesfromathemedissueonSequencesandtopology
EditedbyAnnaPanchenkoandMonikaFuxreiter
https://doi.org/10.1016/j.sbi.2019.03.009
0959-440X/ã2018 Elsevier Ltd. All rights reserved.
Introduction
Manyproteinsharbourextensiveregionsthatlackstable secondary or tertiary structure in their native unbound states[1,2,3].Theseproteinsegments,knownas intrin-sicallydisorderedregions(IDRs),arepredictedto consti-tute up to 40% of the residues in higher eukaryotic proteomes[4,5].Giventhatmanyresiduesintheglobular regionsoftheproteomeareburiedininaccessible stabi-lisinghydrophobiccores,uptohalfofthesurfaceareaof the human proteome accessible for protein interaction maybein unstructuredregions(Figure1a).Decadesof researchhaveuncoveredthekeyfunctionalrolesofthese
regionsin diversebiological systems.Akey findingwas the discovery that IDRs contain interaction modules, such as short linear motifs (SLiMs) and intrinsically disordered domains (IDDs), that mediated interactions andtherebyconferfunctionality[6,7,8].Estimates sug-gest that tens of thousands of interaction modules are encoded in the IDRs of the human proteome[9]. These modulesengage in diversesetsof activities that include providing enzymedockingsites regulating pro-teinmodification states,controlling proteinstability(by recruiting ubiquitin ligases), acting as signals to target proteins to specific subcellular locations, directing dynamic complex formation or driving concentration-dependentphasetransitions[2,6,10].Conversely,IDRs cancontributetoproteinfunctionasadirectresultoftheir structuralpropertieswithouttherequirementofabinding event,forexample,byactingasflexiblelinkersor entro-picchains[11,12].Finally,layersoftranscriptional, post-transcriptional andpost-translational regulationadd cell type and cell state dependent conditionality to these IDR-encodedfunctions[13,14,15,16].
Figure1
58Å2 99Å2
11 million residues 3-4 million residues in IDRs Å2 accessible surface per residue 350 million Å2 in IDRs
Disordered Disordered Disordered Disordered Folded Folded
Dynamic Compact Extended Residual Structure Unstable/Partial Stable
State Transition State
Mechanism
Post-translational modification - Moiety attachment and removal
- Proteolytic cleavage - Peptide isomerization
pH changes Oxidative stress Crowding/Concentration
Interactor binding Allosteric Metal binding Mechanical forces Alternative transcription/splicing
40 60 80 100 120
(a)
(b)
(c) (d)
(e)
(f) (g)
(h)
α β
Current Opinion in Structural Biology
structural changes to regulate a protein’s function (Figure 1c) (Box 1). In order to reassess the structure– function paradigm, the structural biology field must unravel the mechanistic principles that encode such intramolecular interactions and their effects on IDR functions. Furthermore, they must do so in a cellular context where additionalcontributions from physiologi-cally relevant complex association and quinary interac-tions with cellular components may further exacerbate thesestructure–functionrelationships.
Thisperspectivefocusesonthestructuralmodulationof the binding attributes of the functional modules com-monly found in IDRsand highlights openquestionsin the field regarding the link between the structure and interactionsofIDRs.Inrecentyears,anew understand-ingof proteinIDRsaskeyregulators ofbasicbiological functions has emerged. It is vital that we discover the generaldesignprinciplesencodingthestructural proper-tiesofIDRsandtheeffectofthesestructuralproperties onthefunctionofIDRs.However,thiswillrequirenew advancesinkeymethodologiestocomprehensivelystudy theseelusivebiologicalphenomena[20].
The
contribution
of
structure
to
IDR-binding
events
Local—transientstructuralelements
Most bindingevents involving IDRsnecessitate transi-tionsfromdynamicunboundstatestomore constrained bound protein states [8]. Although, many of these
interaction interfaces retain significant flexibility [21]. Furthermore, in some cases, high levels of dynamism canbeobservedevenintheboundstate[22]. Approxi-mately two-thirdsofthefunctional IDRmodules struc-turally solved in complex with their binding partners adopt defined secondary structures when bound [23]. Inthese cases,apreformedboundconformationcanbe arequirementforbinding,aprocessknownas conforma-tional selection. Alternatively, theconformation can be adopteduponbinding,aprocessknownasinducedfit.In manyinstances,bothmechanismscontributetotheactual binding event [24,25,26].For several structurally char-acterised IDRs that interact with globular protein domains via disorder to order transitions, bound IDR statescanbedetectedexperimentallyin freemolecules astransientlypopulated,pre-structuredmotifs(PreSMo) [27]. PreSMo’s exert pronounced effects on actual binding behaviours because they reduce entropy costs associated with folding-upon-binding transitions [28]. Therefore, the sequence of the functional module encodes both the physicochemical complementarity to theIDR-bindinginterfaceandthestructuralpropensity of theregion,andevolutioncantunetheseattributesto optimisebindingattributes[29,30].Anelegantexample to illustrate the contribution of transiently populated structures giving rise to defined binding affinities and productive biologicaloutcomesisthep53–Mdm2 inter-action(Figure1d).Byartificiallyincreasingthelevelsof residual helicity within the IDR PreSMo of p53, the authors generated p53 mutants with enhanced Mdm2-binding affinities [28,31]. Similarly, in the cell, the population of preformed secondary structural elements can also be modulated to strengthen or weaken IDR– ligand interactions in response to external and internal cellularcues(Figure1c).Forexample,phosphorylationof the cleavage and polyadenylation factor Pcf1-binding motif withintheCTDrepeatof RNApolymerase (Pol) IIdoesnotformanyintermolecularcontactsbutenhances binding affinity bystabilising theboundb-turn confor-mation [32,33] (Figure 1e). Another common example is the post-translational modification of the flanks of a-helices to positively or negatively regulate helicity andmodulatebinding[34,35,36].
Regional—compactstates
The accessibilityofafunctionalmodulefor recognition byabinding partnerisafundamentalrequirementof a biomolecular binding event. Non-uniform accessibility resultingfrompreferentiallysampledcompactdisordered states are functionally intriguing. Compact collapsed statescanbedrivenbylocalorlong-rangeintramolecular contacts, with contributions from the physicochemical properties and flexibility of the polypeptide chain [17,37]. Compaction will influence the binding attri-butes to the modules within a region by limiting the accessibility of the functional modules. The functional role of constitutivecollapsed disordered stateshasonly Box1Howisthestructure/functionrelationshipofanIDR
conditionally modulated?
been studied in a handful of proteins. For example, a-synucleinwhereintramolecularcontactspromote com-pactconformationalstatesandshieldtheamyloidogenic NACregion[38].Interestingly,nitrationofC-terminal tyrosines in a-synuclein has been shown to shift the population of sampled conformational states and this changecanmodulatebindingoftheN-terminusto mem-branes[39].Thisexamplerepresentsamodeofallosteric regulationthatresultsnotfromthepropagationof struc-turalchangesthroughaglobularstructure,asobservedin classicalallostery,butresultsfromthemodulationofthe ensemble-averaged contribution of all sampled confor-mationalstates[40].Most of thecomprehensively char-acterisedexamplesofafunctionalroleofaccessibilityin IDR function involve large structural transitions and conditionalfolding(Figure1f–h).Theprototypic exam-pleisthephosphorylation-dependentfoldingof Eukary-otictranslationinitiationfactor4E-bindingprotein2 (4E-BP2) that results in a disorder-to-order transition and buries an eIF4E-binding YxxxxLF motif relieving 4E-BP2-dependent inhibition [41]. Several other similar examplesexistandthediversityoftheobserved mecha-nisms suggests widespread usage of disorder-to-order transitionsin thecell. For example, transitioninitiators ranging from oxidation, in the case of Yeast AP-1-like transcriptionfactor(yAP1) resultingin disulphide bond mediatedfoldingoftwodistantregionsintoafour-helix bundlemaskinganuclearexportsignal(NES) [42],to mechanicalunfolding, in the caseof talin to reveal the vinculin-bindingmotifandpermittingbindingofthe N-terminal vinculin headdomain [43], have been charac-terised.Severalkey questionsremainunanswered: how accessiblearetheunstructuredregionsoftheproteomein vivo[20,44]?Whatproportion of theseregions are par-tiallyorfullycollapsed(compactglobuleormolten glob-ule) [38,45,46]? Or even conditionally folded [19,41,42,47,48]?Howistheaccessibilitymodulated bychangestocellstate?Andmostimportantly,howdoes the accessibility of the modules within these regions modulatetheirfunction?
Global—higher-orderstructure
Many eukaryotic proteins are composed of multiple structurally independent segments organised by inter-regioninteractionstoproducethetopologiesthatdefine the super-tertiary structure of the protein [49]. The literature on higher-order structure of IDRs is sparse. SeveralIDRshavephysiologicallyimportant auto-inhib-ited topologiesthat hide afunctional moduleby intra-molecular interactionwith aglobular region,for exam-ple, the Wiskott–Aldrich syndrome protein (WASP) [50,51]. Additional basic cases exist such as the intra-molecularinteractionsmediatedbytheN-terminalIDR of Colicin-N that protects the unstructured receptor binding region against proteolysis [52] orthe organisa-tion of the intrinsically disordered N-terminus of Src kinase around the SH3 domain [45]. These simple
examples raise the question can multiple disordered regions in a protein orprotein complex have complex higher-order topologies that modulates function [53]? Recent breakthroughs have shown chromatin in the nucleus has spatial organisation [54]. Distinct regions, knownastopologicallyassociatingdomain(TADs), pref-erentially co-localise in functionally significant higher-order chromatin structure. Analogously, long distance intramolecular interactions organising topology associ-ated intrinsically disordered regions (TAIDRs) could result in preferential three-dimensional proximity of distinctdisorderedregions withinaprotein orcomplex (Figure 2a).This higher-order topology could act as a platformforsignalintegrationbyallowingthefunctional modulesintheseregionstocross-talk.Topologiesofthis typehavebeenhypothesisedfortheGabproteinfamily [55],buttodate,no experimentalevidence validating these hypotheses has been published. Hypothetically, the TAIDRs could be organised by numerous mecha-nismsincluding interaction with aglobular region, dis-order-disorder contacts or membrane anchoring and these mechanisms could be conditionally regulated to modulateprotein function.Thisincludesregulationby proteinchaperonessuchas14-3-3orLC8thatcouldact asprotein organisersanalogoustotheCCCTC-binding factor (CTCF)-dependent organisation of chromatin TADs[54,56,57].
Quaternary—IDRsincomplexes
complexes can easily be built using co-operative func-tional modules within multiple IDRs and numerous keyinteractionsinthecellutilisethesedesignprinciples, for example, the high density of SH2 domain-binding motifsinlargesubmembranesignallingcomplexes[61], therepetitiveIDRsdrivingphase transition[66],orthe FG-repeat motifs in the nuclear pore [67]. Conversely, themultimerisationoftheIDR-binding domain-contain-ing protein can also be required for IDR binding. A canonical example is the trimerisation of TRAF2 requiredtobindthreeTRAF2-bindingmotifsintumour necrosisfactorreceptorsignalling[68,69]whereasingle motif-domain interactionisunableto producea biologi-cally relevantinteraction [70].Inmanycases,the oligo-merisationoftheIDR-containingorIDR-bindingprotein can be an important regulatory step, consequently, the system can use these properties to robustly encode a regulatoryoutput [68].
Conclusion
This perspective posits that intramolecular contacts withinIDRswillmodulatethefunctionoftheinteraction interfaces contained within these ubiquitous regions. These contacts will encode the transient secondary structure, accessibilityandthehigher-order structureof
functional module-containing regions to contribute to their binding attributes, and thereby, function. The mechanismscontrollingthestructuralpropertiesofthese regions will be both constitutive and non-constitutive. The structural properties of the constitutive examples will be directly encoded in the primary sequence of the IDR(s) and will fine-tune the binding attributesof thefunctionalmodulesfortheirbindingpartner.Whereas thestructuralpropertiesofthenon-constitutiveexamples will bemodulated in acellstate-dependentmanner by external factors and act as conditional decision-making regions. Given the evolutionary plasticity of IDRs and the functional modules commonly found within them [29,30,71], any regulatory mechanism controlling the function of a disordered region that is possible and accessible toevolutionwilllikelyexist.
The searchforfunctionalmodulesin IDRshas increas-ingly become the focus of intense research and the methodology for the functional analysis of disordered regions has reached the cusp of ahigh-throughput age [72]. Conversely, most of the structural mechanisms modulating IDR function are not easily accessible to thecurrently availablestructuralbiologymethods. This suggests that the paucity of examples of structural Figure2
(a) (b)
(c)
Contact Frequency Crosstalk TAIDR5
TAIDR4
TAIDR3 TAIDR1
TD1
TD2 TD3
TD4 TD5
Binding strength
In vitro In cell
low high
Motif Domain Disorder Contacts
Weak Strong
Disordered Folded Disordered Disordered Disordered
Dynamic Stable Compact Extended Compact
Current Opinion in Structural Biology
regulationoffunctionalmoduleswithinIDRsisa reflec-tionoftheexperimentaldifficultiescharacterisingthese mechanisms(Box2).Consequently, itisimportant that we develop an in vitro, in cell and in silico framework capableofaccessingthesubtleyetfunctionallyimportant contribution of the conformational space sampled by IDRs [20]. With such a framework in place, we can comprehensivelyinvestigatethedesignprinciples encod-ingfunctionalinformationinthedynamicinterconverting conformationsofIDRs.First,weneedtotakeonesimple step:wemuststartthinkingaboutintrinsicallydisordered regionsin threedimensions.
Conflict
of
interest
statement
Nothingdeclared.
Acknowledgements
We apologise to all colleagues whose work could not be cited here owing to spacerestrictions.WewouldliketothankPhilippSelenko,MalvikaSharan andHariArthanariforfruitfuldiscussionsandcriticalevaluationofthe manuscript.Funding:ThisworkwassupportedbyaScienceFoundation Ireland (SFI) Starting Investigator Research Grant [grant numbers 13/SIRG/ 2193].
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