Intrinsically disordered linkers control tethered kinases ... › content › 10.1101 ›...

Dyla&Kjaergaard(2020) www.biophysics.dk-1-

Intrinsicallydisorderedlinkerscontroltetheredkinasesviaeffectiveconcentration

MateuszDyla1,2andMagnusKjaergaard1-4*

1DepartmentofMolecularBiologyandGenetics,AarhusUniversity2TheDanishResearchInstituteforTranslationalNeuroscience(DANDRITE),NordicEMBLPartnershipfor

MolecularMedicine3CenterforProteinsinMemory-PROMEMO,DanishNationalResearchFoundation4AarhusInstituteofAdvancedStudies,AIAS,AarhusUniversity*Correspondingauthor:[email protected]

Abstract:

Kinasespecificityiscrucialtothefidelityofsignallingpathways,yetmanypathwaysuse

thesamekinasestoachievewidelydifferenteffects.Specificityarises inpart fromthe

enzymaticdomain,butalso from thephysical tetheringofkinases to their substrates.

Suchtetheringcanoccurviaproteininteractiondomainsinthekinaseorviaanchoring

and scaffoldingproteins, and candrastically increase the kinetics of phosphorylation.

However,wedonotknowhowsuchintra-complexreactionsdependonthelinkbetween

enzyme and substrate. Here we show that the kinetics of tethered kinases follow a

Michaelis-Menten like dependence on effective concentration. We find that

phosphorylationkineticsscalewiththelengthoftheintrinsicallydisorderedlinkersthat

jointheenzymeandsubstrate,butthatthescalingdiffersbetweensubstrates.Steady-

statekineticscanonlypartiallypredictratesoftetheredreactionsasproductreleasemay

obscure the rate of phospho-transfer. Our results suggest that changes in signalling

complexarchitecturenotonlyenhancetheratesofphosphorylationreactions,butmay

alsoalter therelativesubstrateusage.Thissuggestsamechanismforhowscaffolding

proteinscanallostericallymodifytheoutputfromasignallingpathway.

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 4, 2020. . https://doi.org/10.1101/2020.04.04.023713doi: bioRxiv preprint

https://doi.org/10.1101/2020.04.04.023713


Introduction

Cellular signalling relies on cascades of enzymes such as protein kinases and

phosphatases.Signallingfidelityrequireskinasestousespecificsubstrates,althoughthe

samekinasescantakepartindifferentpathwayswithdifferentsubstrates.Forexample,

proteinkinaseA(PKA)isinvolvedinsignallingpathwaysthroughoutcellbiology.1Kinase

specificity arises in part from the linear motifs surrounding the phospho-accepting

residue.However,invivosubstrateusagecanonlybepredictedpartiallyfromsequences

motifsaskinasesignallingisalsoregulatedthroughthelocalabundanceofenzymes.2The

latterclassofeffectsismuchlessunderstoodquantitatively.

The localabundanceof signallingenzymes is regulatedatmany levels.At thecellular

level,kinasesare targeted toe.g.anorganelleor thecellmembrane.At themolecular

level, enzymes are targeted to a subset of their potential substrates by tethering to

scaffolding proteins. In multidomain enzymes, such tethering occur via protein

interaction domains attached to the catalytic domain.3 Alternatively, enzymes can be

tetheredbyscaffoldingproteins thatbind theenzymeandsubstratesnon-covalently.4

PKA isregulatedbymore than50differentAkinaseanchoringproteins(AKAPs) that

directthekinasetodifferentpathways.5PKAmaydissociatefromitsregulatorysubunit

andthusAKAPsuponactivation,butrecentlyitwasdemonstratedthatactivePKAmay

remaintetheredtoAKAP.6TheintrinsicallydisorderedAKAPsthusrestrictthekinaseto

aregionwhosedimensionsaredefinedbythelengthandcompactnessoftheanchoring

protein.7 Similarly, disordered linkers of variable length regulate the activity of the

dodecamerickinaseCaMKIIbycontrollingthebalancebetweeninter-andintra-subunit

phosphorylation.8Manykinasesignallingreactionsthusoccurinternallyinproteinsorin

proteincomplexes.

Whenakinaseisphysicallyassociatedtoitssubstrate,thetetheredsubstrateisdistinct

fromallother substrates in the reactionmixtureand isusuallyphosphorylatedmuch

faster.Thekineticsofsuchreactionsdifferfromsteady-stateenzymekineticsastethered

catalysisiseffectivelysingleturnover.Duetotheswitch-likefunctionsofmanykinases,

theymayonlyneedtophosphorylateasinglesubstratetoexertabiologicalfunction.9

This canbe seen e.g. in thenegative feedback loop formedbyAKAP79-tetheredPKA,

whichinactivatesadenylatecyclasebyphosphorylation.10Therateofintramolecular(or


https://doi.org/10.1101/2020.04.04.023713


intra-complex) reactions isexpected tobeconcentration independentalthough this is

rarely tested for tethered kinases. Instead, the encounter rate is determined by the

connection between enzyme and substrate. The quantitative description of tethered

catalysisishinderedbythelackofframeworktodescribetheconnectioninkineticterms.

Wepropose thatmany linkers canbe accounted for by the effective concentrationof

substratetheyenforce.Enzymescaffoldingproteinsandinterdomainlinkersinkinases

are often intrinsically disordered as they allow the kinase domain to search for its

substrates.11Forfullydisorderedlinkers,theeffectiveconcentrationscanbeestimated

frompowerlawscalingwiththelengthofthelinker.12Kinasestetheredbydisordered

protein linkers thus offer a chance to understand the general principles that govern

tetheredcatalysis.

Hereweinvestigatehowtheconnectionbetweenakinaseanditssubstrateaffectsthe

phosphorylationkinetics.Weuseamodelsystemconsistingofthecatalyticdomainof

PKAtetheredtoasubstrateviavariableintrinsicallydisorderedlinkers.Weshowthat

phosphorylationratevariesbyseveralordersofmagnitudedependingonthesubstrate

and the linker. We show that phosphorylation rate follows a Michaelis-Menten like

dependence on the effective concentration enforced by the linker, with steady-state

parameterspartiallypredicting theratesof tetheredsingle turnoverphosphorylation.

Thisprovidesabaselineforinterpretinghowsignallingcomplexarchitectureregulates

tetheredenzymes.

Materials and Methods

Preparation of DNA constructs: The plasmid containing the catalytic domain of PKA

(PKAc)wasagiftfromSusanTaylorviaAddgene(#14921).13Theotherplasmidswere

synthesized by Genscript and codon optimized for expression in E. coli. The coding

regionswereclonedintopET15bvectorsusingtheNdeI/XhoIsites.ThedisorderedGS

linkers from the linker library generated previously12 were cloned into the protein

constructsusinguniqueNheI/KpnIsites.Thesequencesofproteinconstructsaregiven

intheSI.

Proteinexpressionandpurification:Proteinconstructscontainingthecatalyticdomainof

PKA linked by variable length linkers to the MBD2 coiled-coil domain (MBD2-(GS)n-


https://doi.org/10.1101/2020.04.04.023713


PKAc)wereexpressedinC41(DE3)cellsinLBmediumwith100µg/mLampicillinat37°C

andshakingat120RPM.Thecultureswereinducedwith1mMIPTGatOD600=0.6-0.8

andthetemperaturewasdecreasedto30°Cforovernightexpression.Proteinconstructs

containingPKAsubstrateslinkedtothep66αcoiled-coildomain(p66α-(GS)n-substrate)

wereexpressed inBL21(DE3) cells in ZYM-5052auto-inductionmedium14 containing

100µg/mLampicillinat37°Candshakingat120RPMovernight.

The cells were harvested by centrifugation (15 min, 6000 g), bacterial pellets were

resuspendedinbindingbuffer(20mMNaH2PO4,0.5MNaCl,5mMimidazole,0.1mM

TCEP,0.2mMPMSF,50mg/Lofleupeptin,50mg/Lpepstatin,50mg/Lchymostatin,pH

7.4)andlysedbysonication(50%dutycycle,maximumpowerof75%,sonicationtime

of6min).Thelysatewascentrifuged(20min,27000g)andthesupernatantwasapplied

to gravity flowcolumnspackedwithNi-NTASuperflow (QIAGEN).The columnswere

washedwithbufferscontainingincreasingconcentrationsofimidazole(20-50mM)and

wereelutedwithelutionbuffer(20mMNaH2PO4,0.5MNaCl,500mMimidazole,0.1mM

TCEP).ElutedproteinsamplescontainingPKAsubstratesweredialyzedagainstbinding

bufferwithoutimidazoleovernight,followedbycleavageofHis-tagbythrombin(1:1000

thrombintosubstrateratio)at37°Candshakingat300RPMfor30min.Cleavedsamples

wereappliedtogravityflowcolumnspackedwithNi-NTASuperflow(QIAGEN)andflow-

throughwascollected.Allproteinsampleswereadditionallypurifiedbysize-exclusion

chromatography(SEC)inTBSbuffer(20mMTris-base,150mMNaCl,0.1mMTCEP,pH

7.6)usingSuperdex200IncreaseandSuperdex75Increasecolumns(GEhealthcare)to

purifyPKAcanditssubstrates,respectively.

Quench-flow measurements: Single-turnover kinetic measurements were performed

usingaKinTekCorporationRQF-3Rapidquench-flowinstrumentat30°C.MBD2-(GS)n-

PKAc and p66α-(GS)n-substrate were diluted to 10x final concentration in enzyme

dilutionbuffer(50mMTris-base,0.1mMEGTA,1mg/mlBSA,1mMTCEP,pH7.6)and

TBS (20mMTris-base,150mMNaCl,pH7.6), respectively.Quench flowexperiments

were executed by loading the catalytic domain of PKA and its substrates (final

concentration0.5µMunlessindicatedotherwise)intoonesampleloopand[γ-32P]ATP

(finalconcentration0.1mMof100-200countsperminute(c.p.m.)pmol-1)intotheother.

Phosphorylationreactionswerecarriedoutinthereactionbuffercomposedof50mM


https://doi.org/10.1101/2020.04.04.023713


Tris-base,0.1mMEGTA,10mMmagnesiumacetate,150mMNaCl,pH7.6.Thereactions

werequenchedusing150mMphosphoric acidusing the “constantquench”option to

limit the total quenched reaction volume to 70 µL. Phosphorylated substrates were

separated from unreacted ATP by spotting the quenched reactions onto P81

phosphocellulosedisks(JonOakhill,St.VincentInstitute,Melbourne).Thefilterswere

washed3timeswith75mMphosphoricacid,rinsedwithacetone,dried,andcountedon

the32Pchannelinscintillationcounterasc.p.m.Controlexperimentswereperformedto

determinebackgroundphosphorylationlevelofsubstratemotifsintheabsenceofPKAc,

aswellasautophosphorylationofPKAcintheabsenceofsubstrates.Backgroundvalues

(typicallynegligible)weresubtractedfromtherawdata.Specificphosphorylationwas

determinedasamountof32P-incorporatedsubstrate(pmol)basedon32P-ATPstandard

curve,wherec.p.m.valuesofknownamountsofspiked32P-ATPweremeasured.

Steady-statekinetics:PKAc(withouttheMBD2dimerizationdomain)wasdilutedto10x

finalconcentrationinenzymedilutionbuffer(50mMTris-base,0.1mMEGTA,1mg/ml

BSA,1mMTCEP,pH7.6)andp66α-(GS)n-substratewasdilutedto5xfinalconcentration

inTBS(20mMTris-base,150mMNaCl,pH7.6).Steady-stateexperimentswereexecuted

bymanualadditionof[γ-32P]ATP(finalconcentration0.1mMof100-200c.p.m.pmol-1)

into reactionmixcontaining1nMPKAcandp66α-(GS)n-substrate (final concentration

10-1820µM)inareactionbuffer(50mMTris-base,0.1mMEGTA,10mMmagnesium

acetate,150mMNaCl,pH7.6)at30°C.Everyminute5µlofthereactionmixwasspotted

ontoaP81filterdiskandplacedinto75mMphosphoricacidtoquenchthereaction.The

filterswerewashed3timeswith75mMphosphoricacid,rinsedwithacetone,dried,and

countedonthe32Pchannel inscintillationcounterasc.p.m.Controlexperimentswere

performedinthesamewayasinquench-flowexperiments,andspecificphosphorylation

was determined likewise. Initial velocities at different substrate concentrationswere

derived froma slopeof a linear regressionaspmolof 32P-incorporated substrateper

minute.ThedatawerethenfittedbyaMichaelis-Mentenmodel,andkcatwascalculated

bydividingVmaxby0.005pmolofPKAcandby60(min/s).

Results

To probe the linker dependence of tethered catalysis, we designed a model system

composedofthecatalyticdomainofproteinkinaseA(PKAc)tetheredtoitssubstratevia


https://doi.org/10.1101/2020.04.04.023713


disorderedlinkers(Fig.1A,S1).WeusedtheoptimalPKAsubstrateKemptide(WTinthe

following)16andvariedsubstratequalitybymutagenesis.Thekinaseandsubstratewere

made as separate proteins and joined non-covalently using heterodimeric coiled-coil

domains.Weusetheanti-parallelcoiled-coilformedbetweenp66αandMBD215asithas

lownMaffinity,andisstableonthetimescaleofthetetheredreaction.Thecoiled-coil

domains were joined to the PKAc/substrate by an exchangeable linker flanked by

restriction sites compatible with our library of disordered linkers.12 To approximate

passive,entropicspacers,weusedglycine-serinerepeatlinkerscontaining2,20,60and

120residues.

Figure1:Areductionistmodelsystemfortetheredphosphorylation.(A)ThecatalyticdomainofPKAanditsconsensussubstratemotifarejoinedtothecoiled-coildomainfromMBD2andp66α,respectively.MBD2andp66αformananti-parallelheterodimerwithnanomolaraffinity.15Substratequalitywasvariedbymutationawayfromtheconsensusmotif,andthelinkerwasvariedbyincludingGS-repeatsofvariablelength.(B)Quench-flowwasusedtomeasuresingle-turnoverkineticsbyfollowingincorporationof32Pintothesubstrate.Tetheringofenzymeusingcombinedlinkerlengthof40residuesincreasedtherateby~300-fold at a substrate and enzyme concentrations of 0.5 µM. (C) The rate of the tethered catalysisdecreasedslightlywithincreasingconcentrationofsubstrateandenzyme.

We followed single-turnover phosphorylation in an equimolar mix of kinase and

substrateusingquench-flow.Thereactionwasstartedbyadding[γ-32P]ATP,quenched

byphosphoricacidatreactiontimesfrom2.5msto330s,andfollowedfromtheamount

of32Pinthesubstrate.Initially,wecomparedidenticaltetheredanduntetheredreactions,

wheretetheringincreasedreactionratebymorethan100-fold(Fig.1B).Intra-complex

reactions are concentration independent, whereas the rate of bimolecular reactions

increaseswithconcentration.Therefore,wetestedhowtheconcentrationoftheenzyme

andsubstrateaffected thephosphorylationrate.WhileremainingbelowtheKMof the

substrate, the concentration was increased 10-fold, which led to a slightly slower


https://doi.org/10.1101/2020.04.04.023713


phosphorylation(Fig.1C).Theseobservationssuggestthatphosphorylationoccursintra-

complexwithnegligiblecontributionsfromtrans-phosphorylation.

Totesthowlinkersaffectphosphorylation,wevariedthelengthsoftheGSrepeats.By

combiningkinaseandsubstratevariants,wecreated6complexeswithacombinedlinker

lengthspanningfrom20to180disorderedresiduesinadditiontothe17residuesfrom

restriction sites and the short extra linker in the substrate. For all variants the

phosphorylationreactioncouldbedescribedwellwithasingleexponentialmodel,where

thephosphorylationratedecreasedmonotonicallywithincreasedlinkerlength(Fig.2A,

S2). This suggests that kinase linkers and anchoring proteins directly regulate intra-

complexkinasereactions.

Next, we tested whether substrate quality affects the linker dependence of

phosphorylation.PKArecognizespositiveresiduesinpositions-2and-3relativetothe

phosphorylationsitewithapreferenceforarginineoverlysine.16Toweakenthesite,we

mutatedeachofthetwoarginineresidues inthesubstratemotif to lysineresulting in

variantsR-2KandR-3K.Thephosphorylationratedecreasedmonotonicallywithlinker

lengthforbothmutants,althoughwithdifferentmagnitudes(Fig.2B).Phosphorylation

oftheWTsubstrateonlyslows2-foldfromtheshortesttothelongestlinkers,whereas

theR-3Ksubstrateslowsbymorethan10-fold.Thissuggeststhatsubstratesareaffected

differentlyby tethering, and that thepropertiesof the linkerandsubstrate shouldbe

consideredtogether.


https://doi.org/10.1101/2020.04.04.023713


Figure2:Linkerdependenceofsingle-turnoverphosphorylationrates.(A)Quench-flowkineticsofthephosphorylationreactionsoftheR-3Ksubstratewithdifferentlinkerlengths.ThelinkerlengthisthecombinednumberofGS-repeats in the two linkers. (B)Theobserved rateofphosphorylationof threedifferentsubstratesasafunctionofthecombinedlinkerlength.(C-E)Phosphorylationratesasafunctionoftheestimatedeffectiveconcentrationenforcedbythelinker.EffectiveconcentrationsareestimatedfromthepowerlawmeasuredpreviouslyforGS-linkers12assumingthatthepresenceofthecoiled-coildomainisnegligible.(F)KineticschemeofthetetheredsystemundersaturatingconcentrationofATP.O·ATP–openstateofthetetheredsystem,ATP-bound;C·ATP–closedcatalyticallycompetentstate,ATP-bound;CP·ADP – closed phosphorylated state, ADP-bound; OP – open, nucleotide-free state. Nucleotide andphosphorylationareshownasorangemoietiesinthecartoon.

TheattenuatedlinkerdependenceoftheWTsubstratecouldinprinciplebecausedby

ATP binding becoming the limiting factor at high phosphorylation rates. To test this

possibility,wecarriedoutquenchflowexperimentsata1mMATPinsteadof100µM.

Theamountof[γ-32P]ATPwaskeptconstanttostaywithinpermittedradioactivitylevels,

andsoweincreasedtheproteinconcentrationto5µM,whichhadaminimaleffecton

kinetics (Fig. 1C), tomaintain a robust signal. For theWT substrate, phosphorylation

rates increased by ~30% (Fig. S3) for all linker combinations. A uniform rate


https://doi.org/10.1101/2020.04.04.023713


enhancementat1mMATPisexpectedifthekinaseisnotfullysaturatedwithATPat100

µM.PreviousstudiesreportaKMforATPof10µMatdifferentbuffercondition,17sothe

kinasemaynotbefullysaturatedat100µM.Totestthisexplanation,wealsotesteda

slowerR-3Kvariantwith1mMATP,andsawasimilarincreaseintheobservedrate(Fig.

S2)consistentwithanincreaseinlevelofboundATP.Thissuggeststhattheattenuated

linkerdependencefortheWTisnotduetoATPbindingbecomingkineticallylimiting,but

ratherrepresentssaturationofthetetheredsubstrate.

Tetheredreactionsareexpectedtofollowfirstorderkineticswiththecontactfrequency

of reactants determinedby their physical connection.18 The contact frequency canbe

expressed as an effective concentration, which in our case corresponds to the

concentrationoffreesubstratethatwouldencountertheenzymeasoftenasthetethered

substrate. Such effective concentration can occasionally bemeasured by competition

experiments,12,19,20althoughinmostproteinshastobeestimatedtheoretically.21–23Our

group previouslymeasured the scaling of effective concentrationswith the length of

disordered linkers including the sequences used here.12 To estimate the effective

concentration of the tethered substrate, we used the power law determined for GS-

linkers previously, and plotted phosphorylation rates as a function of effective

concentration (Fig.2C-E).The tether inourkinase-substrate systemalso includes the

coiled-coil domain, which act as a rigid link in the flexible linker. Previous effective

concentration measurements suggest a similar scaling with linker length for such a

system,sowedisregardthecontributionofthecoiled-coildomainalthoughitpotentially

introducesasystematicerror.19

Thephosphorylationratesaturatedathigheffectiveconcentrationforallvariants.The

WT substrate was close to saturation already at the concentrations enforced by the

longest linkers. Incontrast,R-2Konlysaturatedat thehighesteffectiveconcentration

enforced by the shortest linkers, whereas R-3K has not plateaued yet at the highest

effectiveconcentration.Themaximalratealsodifferedstronglybetweenthesubstrates,

astheR-2KandR-3KvariantssaturatedatmuchlowerratesthantheWTsubstrate.In

total, this suggests that the single turnover reaction canbedescribedby aMichaelis-

Mentenlikedependenceontheeffectiveconcentrationwithahalfwaysaturationpoint


https://doi.org/10.1101/2020.04.04.023713


andmaximal turnover characteristic of each substrate,wherektet,max is themaximum

phosphorylationrateandCeff,50isaneffectiveconcentrationathalf-saturation:

𝑘"#" =𝑘"#",&'(𝐶#**𝐶#**,+, + 𝐶#**

(1)

Todescribethekineticsoftetheredphosphorylationreactions,wederivedrateequations

(SItext)basedonaproduct-releaselimitingmodelforPKA(Figure2F).17,24Underthe

assumptionthatphosphorylation(k2)andproductrelease(k3)areirreversibleandthe

closed,catalyticallycompetentcomplex(C)isinarapidequilibriumwithanopenform

(O), the rate of phosphorylation in the tethered system (ktet) follows the following

relationshiponeffectiveconcentration(Ceff):

𝑘"#" =𝑘2𝐶#**𝐾4 + 𝐶#**

(2)

The rate of phosphotransfer (k2) is directly obtained from the maximum rate of

phosphorylation in the tetheredsystem(ktet,max) andKd isobtained fromaneffective

concentrationathalf-saturationCeff,50,basedonthefitofexperimentaldatatoEquation

1. The fittingparameters are given inTable 1.Kd determined in thisway for theWT

substrateisequalto142µM,similartotheliteraturevalueof200µM17,anditincreased

to837µMand8400µMforR-2KandR-3K,respectively.

Table1:Kineticparametersobtainedfromfitsofexperimentaldata

WT 1mM ATP

WT R-2K R-3K

Tethered ktet,max (s-1) 415 ± 47 307 ± 21 40.0 ± 3.5 = 14.1

Ceff,50 (µM) 107 ± 59 142 ± 42 837 ± 150 8400 ± 910

Untethered kcat (s-1) 37.3 ± 0.8 33.5 ± 0.4 14.1 ± 0.3

KM (µM) 24.0 ± 2.1 200 ± 5.2 973 ± 43

To test whether the rates of tethered catalysis can be predicted from steady-state

kinetics, we recorded a matching untethered steady-state enzyme kinetics for each

substrate(Fig.3).TheKMoftheuntetheredreactionincreasedfrom24.0µMintheWT

substrateto200and973µMinR-2KandR-3K,respectively.KMvaluesforallsubstrates

are an order of magnitude below the corresponding Kd values. To quantify the


https://doi.org/10.1101/2020.04.04.023713


relationshipbetweenKMandKd,wederivedsteady-stateparametersasafunctionofrate

constantsinuntetheredsystem(SItext),whichresultedinthefollowingequation:

𝐾6 = 𝐾4𝑘7

𝑘2 + 𝑘7(3)

KMisexpectedtobesmallerthanKdwhenthesteady-statereactionislimitedbyproduct

dissociation(k3<k2).Inturn,thissuggeststetheredanduntetheredphosphorylationwill

have different half saturation points, as kinases are often limited by product

dissociation.25,26

Figure3.QuantifyingthequalityofPKAsubstratevariants.(A)Rawdatafromasteady-statekineticexperimentperformedat1nMPKAcandanindicatedconcentrationoftheR-2Ksubstrate.Theinsetshowsaschematiccartoonoftheexperiment.(B)Steady-statekineticdataof3substratesfittedbyaMichaelis-Mentenmodel.

Thekcatvaluesdecreasedslightlyfrom37.3s-1inWTto33.5s-1and14.1s-1inR-2Kand

R-3K, respectively. In contrast, there was a big difference in ktet,max between the

substrates:itwas8-foldfasterthankcatforWT(307s-1),roughlythesameforR-2K(40.0

s-1) and seemed to be slower (~3 s-1) for R-3K. As ktet,max corresponds to the rate of

phosphotransferitcannotbelowerthankcat.TheinconsistencyforR-3Kislikelydueto

thepoorlydefinedplateauinthedatafortetheredR-3K,andthusweconstrainedktet,max

toitslowerlimitequaltokcatuponfittingoftheR-3Kdata.Totesthowreactionswith

widelydifferentratesofphosphotransfercanconvergeonsimilarsteady-stateturnover

rates, we simulated kcat as a function of ktet,max for different values of the product

dissociationrateconstant(Fig.4A)asbelow:


https://doi.org/10.1101/2020.04.04.023713


𝑘9'" =𝑘2𝑘7𝑘2 + 𝑘7

(4)

Fig.4Ashowsthatkcatislargelyindependentoftherateofphospho-transfer,whenthe

steady-statereactionislimitedbyproductdissociation.Theobservedvaluesofkcatcan

thus roughly be explained assuming the same rate of product dissociation. Product

dissociationrateswillbesimilar ifADPdissociation is limiting,butmaydiffer for the

peptides,soaperfectfitisnotexpected.Crucially,however,Fig.4Ashowsthatsteady-

statekineticsonlyhavealimitedabilitytopredicttherateofatetheredreaction,andby

extension it cannot predict the relative substrate usage in signalling events that only

requireasingleintra-complexphosphorylation.

Figure4.Simulatedpredictionsfortetheredcatalysis(A)Simulationsofkcatfromktet,maxandk3basedonEquation4inaproductreleaselimitedreaction.Athighktet,maxvalues,kcatisdecoupledfromatherateofthetetheredreactionandmaydisguiselargedifferencesinthetetheredphosphorylationrate.(B)FittingcurvesfromFigure2C(WT)and2E(R-3K)showunequalincreaseinktet(2.1xand9.0x,respectively)uponincreasingCefffrom100to1000µM.

Discussion

Wehavedevelopedareductionisticmodelforphosphorylationbyatetheredkinasethat

mimicskeyfeaturesofkinasesthatcontaindisorderedlinkersorbindflexiblescaffolding

proteins.Themodelsystemomitsthecomplexityofnaturalsignallingcomplexes,butin

returnallowssystematicvariationofthelinkerarchitectureandsubstrate.Thisallowed


https://doi.org/10.1101/2020.04.04.023713


ustodemonstratethatphosphorylationcanbeenhanceddramaticallybytethering,and

this enhancement depends strongly on the linker architecture. We have shown that

single-turnovertetheredphosphorylationcanbedescribedbyaMichaelis-Mentenlike

dependence on the effective concentration, but that the kinetic parameters can only

partiallybedescribedfromsteady-statekinetics.

We have shown that the rate of tethered phosphorylation reaches a plateau at high

effectiveconcentrations,butissuchsaturationrelevantforkinasereactionsinnatural

signalling complexes? Saturation occurs when the effective substrate concentration

exceedstheaffinityofthekinase.Theaffinityofkinasesforshortsubstratemotifscan

typicallyreachlowµM values,althoughmanybiologicallyimportantsubstratesarefar

fromtheoptimalsequencemotifandmayhaveevenloweraffinities.Modelsystemsand

computationalmodellingsuggestthattheeffectiveconcentrationsinproteincomplexes

reach the lowmM range.12,20,27 This suggests that signalling complexes can reach the

saturation regime, where e.g. the shortening of a linker will no longer enhance the

reaction.

Saturationathigheffectiveconcentrationsprovidesamechanismforhowchangesinthe

linkerarchitecturecanchangetherelativerateoftetheredsubstrates,andthusalterthe

substrateusageofakinase.Forexample,considertwoadjacentphosphorylationsites

corresponding to the WT and R-3K substrates tethered at the same effective

concentration(Fig.4B).Ifthescaffoldingproteinchangesstructureduetoe.g.effector

bindingoralternativesplicing,theeffectiveconcentrationmaychangefrom100µMto1

mM, corresponding to GS-linkers of 257 and 53 residues, respectively. These values

roughlymatchthedifferenceinlinkersbetweenthea andb-isoformsofCaMKII.28Asthe

WTsubstrateisnearsaturation,thephosphorylationrateofR-3Kwillbeenhanced4.3-

fold more. This suggests that changes in linker architecture that increase effective

concentrationsshiftrelativesubstrateusagetowardslowaffinitysubstrates.

Tetheredkinasesareoftenpartofswitch-likefunctions,wherethebiologicaleffectonly

requiresphosphorylationof fewsubstrates.Examples includeautophosphorylationof

kinases such as CaMKII or activation by transphosphorylation of receptor tyrosine

kinases.Furthermore,activatorystimuliareoftentransientsuchase.g.thebriefburstof


https://doi.org/10.1101/2020.04.04.023713


Ca2+ triggered by opening Ca2+-channels. In such switches, the number of substrates

processedpertimeislessimportantthanthetimerequiredforthefirstphosphorylation.

Yet steady-state kinetics remains themainmodeof evaluating substrates, even if the

nativephysiologicalcontextisatetheredcomplex.Weshowthatsteady-stateparameters

candisguisebigdifferencesintherateoftetheredphosphorylation:TheWTandR-2K

substrateshada8-folddifferenceinktet,maxeventhoughthekcatvaluesaresimilar.This

occurs because product dissociation limits the steady-state reaction, but not the

phosphorylationof tetheredsubstrate.The latter ismorerelevant to tetheredkinases

withswitch-likefunctions,whichsuggeststhatsubstratepropertiesshouldbeevaluated

insingleturnoverexperiments.

Kinasesareoftentetheredtosubstratesvianon-covalent interactions,whichsuggests

that dissociation and rebinding should be considered. Themodel proposedhere only

considersastaticconnection,eventhoughthefitteddatausesanon-covalentconnection

betweenkinaseandsubstrate.Wesuggestthattheinteractioncanbeapproximatedas

static,whenitslifetimeismuchlongerthanthecatalyticcycle.Assuchourmodelsystem

describeshigh-affinityscaffoldinginteractionsinadditiontolinkersthataretrulystatic,

such as in kinase auto-phosphorylation reactions. For weaker interactions, single-

turnover and steady-state kineticmodels start toblend andwill accordinglybemore

complex.Webelievethatstaticmodelwillprovideabaselineforsuchefforts.

Duringthepreparationofthismanuscript,apre-printappearedwithsimilaraims,but

verydifferentresults.29Inagreementwithourresults,SpeltzandZalatanfoundthatfor

tethered PKAc shorter linkers increased the rate of phosphorylation. Despite the

similaritybetweenthemodelsystemsused,thereactionoccurredinadifferentkinetic

regimewithamaximalktetof~0.002s-1.Theunexpectedlyslowphosphorylationrate

was explained by effective concentrations of ~80 nM, which is 50.000-fold below

predictionfromasimplegeometricmodel.29Thesystemsdiffermainlyinthesubstrate

andthelinkersused.Itispossible,thatthekineticssimplyreflectthedifferentsubstrates,

assubtledifferencesinthemotifmayhaveabigeffectontherateofthetetheredreaction.

Additionally, Speltz and Zalatan use short linkers that also contain a folded domain.

Likely,thediscrepancybetweenexperimentalandpredictedvaluesshowsthelimitation

ofsimplepolymermodels forshort linkers.Suchsystemscaneasilybecomesterically


https://doi.org/10.1101/2020.04.04.023713


limitedbytheexcludedvolumefromfoldeddomainsandsequence-specificinteractions

that lead to internal friction. In contrast, long disordered linkers allow almost free

orientation of domains,30 and are likely to provide the most robust architecture for

engineeredscaffoldingproteins.

Tetheredreactionsarenotjustimportantforkinases.Tetheringisarguablyevenmore

importantforphosphatasesasthesubstratemotifcontributeslittlespecificity.Instead,

phosphatasesareregulatedbyanchoringproteins,ande.g.proteinphosphatase-1hasat

least 200 different regulatory subunits that target it to different substrates.31 These

anchoringinteractionslikelydisplayasimilardependenceforlinkerarchitectureasseen

here.Tetheringisalsoincreasinglyusedasatherapeuticinterventionstrategy.Enzymes

tetheringisimportantine.g.PROTACsthattargetproteinsfordegradationbytethering

themtoubiquitinligase.32Tetheringhasalsobeenusedpharmacologicallytoenhance

otherclassesofenzymesincludinge.g.proteases33andphosphatases.34Suchconnector

moleculeswilllikelydisplayasimilardependenceonconnectiontothesystemsstudied

here, and mechanistic studies of tethered catalysis is thus both of fundamental

importance to understand signalling pathways and to optimize pharmaceutical

interventionstrategies.

Acknowledgments

ThisworkwassupportedbygrantstoM.K.fromthe“YoungInvestigatorProgram”ofthe

Villum Foundation, the AIAS COFUND program funded by the EU FP7 programme

(Agreementno.754513)andPROMEMO–Center forProteins inMemory,aCenterof

Excellence funded by the Danish National Research Foundation (grant number

DNRF133). We would like to thank XavierWarnet and Sujata Mahapatra for critical

commentstothemanuscript.

References

(1)Taskén,K.,andAandahl,E.M.(2004)LocalizedEffectsofcAMPMediatedbyDistinct

RoutesofProteinKinaseA.Physiol.Rev.84,137–167.

(2)Miller,C.J.,andTurk,B.E.(2018)Homingin:MechanismsofSubstrateTargetingby

ProteinKinases.TrendsBiochem.Sci.43,380–394.


https://doi.org/10.1101/2020.04.04.023713


(3)Pawson,T.(1995)Proteinmodulesandsignallingnetworks.Nature373,573–580.

(4)Good,M.C.,Zalatan,J.G.,andLim,W.A.(2011)ScaffoldProteins:Hubsfor

ControllingtheFlowofCellularInformation.Science(80-.).332,680–686.

(5)Wong,W.,andScott,J.D.(2004)AKAPsignallingcomplexes:focalpointsinspace

andtime.Nat.Rev.Mol.CellBiol.5,959–970.

(6)Smith,F.D.,Esseltine,J.L.,Nygren,P.J.,Veesler,D.,Byrne,D.P.,Vonderach,M.,

Strashnov,I.,Eyers,C.E.,Eyers,P.A.,Langeberg,L.K.,andScott,J.D.(2017)Local

proteinkinaseAactionproceedsthroughintactholoenzymes.Science(80-.).356,1288–

1293.

(7)Smith,F.D.,Reichow,S.L.,Esseltine,J.L.,Shi,D.,Langeberg,L.K.,Scott,J.D.,and

Gonen,T.(2013)IntrinsicdisorderwithinanAKAP-proteinkinaseAcomplexguides

localsubstratephosphorylation.Elife2,e01319.

(8)Bhattacharyya,M.,Lee,Y.K.,Muratcioglu,S.,Qiu,B.,Nyayapati,P.,Schulman,H.,

Groves,J.,andKuriyan,J.(2020)FlexiblelinkersinCaMKIIcontrolthebalancebetween

activatingandinhibitoryautophosphorylation.Elife9,e53670.

(9)Taylor,S.S.,Ilouz,R.,Zhang,P.,andKornev,A.P.(2012)Assemblyofallosteric

macromolecularswitches:LessonsfromPKA.Nat.Rev.Mol.CellBiol.13,646–658.

(10)Bauman,A.L.,Soughayer,J.,Nguyen,B.T.,Willoughby,D.,Carnegie,G.K.,Wong,W.,

Hoshi,N.,Langeberg,L.K.,Cooper,D.M.F.,Dessauer,C.W.,andScott,J.D.(2006)

DynamicRegulationofcAMPSynthesisthroughAnchoredPKA-AdenylylCyclaseV/VI

Complexes.Mol.Cell23,925–931.

(11)Cortese,M.S.,Uversky,V.N.,andKeithDunker,A.(2008)Intrinsicdisorderin

scaffoldproteins:Gettingmorefromless.Prog.Biophys.Mol.Biol.98,85–106.

(12)Sørensen,C.S.,andKjaergaard,M.(2019)Effectiveconcentrationsenforcedby

intrinsicallydisorderedlinkersaregovernedbypolymerphysics.Proc.Natl.Acad.Sci.

116,23124–23101.

(13)Narayana,N.,Cox,S.,Shaltiel,S.,Taylor,S.S.,andXuong,N.H.(1997)Crystal

structureofapolyhistidine-taggedrecombinantcatalyticsubunitofcamp-dependent

proteinkinasecomplexedwiththepeptideinhibitorPKI(5-24)andadenosine.

Biochemistry36,4438–4448.

(14)Studier,F.W.(2005)Proteinproductionbyauto-inductioninhigh-densityshaking

cultures.ProteinExpr.Purif.41,207–234.

(15)Gnanapragasam,M.N.,Scarsdale,J.N.,Amaya,M.L.,Webb,H.D.,Desai,M.a,


https://doi.org/10.1101/2020.04.04.023713


Walavalkar,N.M.,Wang,S.Z.,ZuZhu,S.,Ginder,G.D.,andWilliams,D.C.(2011)

p66Alpha-MBD2coiled-coilinteractionandrecruitmentofMi-2arecriticalforglobin

genesilencingbytheMBD2-NuRDcomplex.Proc.Natl.Acad.Sci.U.S.A.108,7487–92.

(16)Kemp,B.E.,Graves,D.J.,Benjamini,E.,andKrebs,E.G.(1977)RoleofMultiple

BasicResiduesinDeterminingthesubstratespecificityoftheSubstrateSpecificityof

CyclicProteinKinase.J.Biol.Chem.252,4888–4894.

(17)Grant,B.D.,andAdams,J.A.(1996)Pre-steady-statekineticanalysisofcAMP-

dependentproteinkinaseusingrapidquenchflowtechniques.Biochemistry35,2022–

2029.

(18)VanValen,D.,Haataja,M.,andPhillips,R.(2009)Biochemistryonaleash:Theroles

oftetherlengthandgeometryinsignalintegrationproteins.Biophys.J.96,1275–1292.

(19)Sørensen,C.S.,Jendroszek,A.,andKjaergaard,M.(2019)LinkerDependenceof

AvidityinMultivalentInteractionsbetweenDisorderedProteins.J.Mol.Biol.431,4784–

4795.

(20)Mack,E.T.,Snyder,P.W.,Perez-Castillejos,R.,Bilgicer,B.,Moustakas,D.T.,Butte,

M.J.,andWhitesides,G.M.(2012)Dependenceofavidityonlinkerlengthforabivalent

ligand-bivalentreceptormodelsystem.J.Am.Chem.Soc.134,333–345.

(21)Diestler,D.J.,andKnapp,E.W.(2008)Statisticalmechanicsofthestabilityof

multivalentligand-receptorcomplexes.Phys.Rev.Lett.100,178101.

(22)Borcherds,W.,Becker,A.,Chen,L.,Chen,J.,Chemes,L.B.,andDaughdrill,G.W.

(2017)OptimalAffinityEnhancementbyaConservedFlexibleLinkerControlsp53

MimicryinMdmX.Biophys.J.112,2038–2042.

(23)Sherry,K.P.,Das,R.K.,Pappu,R.V.,andBarrick,D.(2017)Controlof

transcriptionalactivitybydesignofchargepatterningintheintrinsicallydisordered

RAMregionoftheNotchreceptor.Proc.Natl.Acad.Sci.114,E9243–E9252.

(24)Adams,J.A.(2001)Kineticandcatalyticmechanismsofproteinkinases.Chem.Rev.

101,2271–2290.

(25)Cook,P.F.,Neville,M.E.,Vrana,K.E.,ThomasHartl,F.,andRoskoski,R.(1982)

AdenosineCyclic3′,5′-MonophosphateDependentProteinKinase:KineticMechanism

fortheBovineSkeletalMuscleCatalyticSubunit.Biochemistry21,5794–5799.

(26)Whitehouse,S.,andWalsh,D.A.(1983)MgATP2--dependentInteractionofthe

InhibitorProteinofthecAMP-dependentProteinKinasewiththeCatalyticSubunit.J.

Biol.Chem.258,3682–3692.


https://doi.org/10.1101/2020.04.04.023713


(27)Sørensen,C.S.,Jendroszek,A.,andKjaergaard,M.(2019)LinkerDependenceof

AvidityinMultivalentInteractionsbetweenDisorderedProteins.J.Mol.Biol.431,4784–

4795.

(28)Sloutsky,R.,Dziedzic,N.,Dunn,M.J.,Bates,R.M.,Torres-Ocampo,A.P.,Page,B.,

Weeks,J.G.,andStratton,M.M.(2019)HeterogeneityinhumanhippocampalCaMKII

transcriptsrevealsanallostericroleforthehubdomaininactivityregulation.bioRxiv

721589.

(29)Speltz,E.B.,andZalatan,J.G.(2020)Therelationshipbetweeneffectivemolarity

andaffinitygovernsrateenhancementsintetheredkinase-substratereactions1–28.

(30)Mittal,A.,Holehouse,A.S.,Cohan,M.C.,andPappu,R.V.(2018)Sequence-to-

ConformationRelationshipsofDisorderedRegionsTetheredtoFoldedDomainsof

Proteins.J.Mol.Biol.430,2403–2421.

(31)Bollen,M.,Peti,W.,Ragusa,M.J.,andBeullens,M.(2010)TheextendedPP1toolkit:

Designedtocreatespecificity.TrendsBiochem.Sci.35,450–458.

(32)Paiva,S.L.,andCrews,C.M.(2019)Targetedproteindegradation:elementsof

PROTACdesign.Curr.Opin.Chem.Biol.50,111–119.

(33)Kitazawa,T.,Igawa,T.,Sampei,Z.,Muto,A.,Kojima,T.,Soeda,T.,Yoshihashi,K.,

Okuyama-Nishida,Y.,Saito,H.,Tsunoda,H.,Suzuki,T.,Adachi,H.,Miyazaki,T.,Ishii,S.,

Kamata-Sakurai,M.,Iida,T.,Harada,A.,Esaki,K.,Funaki,M.,Moriyama,C.,Tanaka,E.,

Kikuchi,Y.,Wakabayashi,T.,Wada,M.,Goto,M.,Toyoda,T.,Ueyama,A.,Suzuki,S.,

Haraya,K.,Tachibana,T.,Kawabe,Y.,Shima,M.,Yoshioka,A.,andHattori,K.(2012)A

bispecificantibodytofactorsIXaandXrestoresfactorVIIIhemostaticactivityina

hemophiliaAmodel.Nat.Med.18,1570–1574.

(34)Yamazoe,S.,Tom,J.,Fu,Y.,Wu,W.,Zeng,L.,Sun,C.,Liu,Q.,Lin,J.,Lin,K.,

Fairbrother,W.J.,andStaben,S.T.(2020)HeterobifunctionalMoleculesInduce

DephosphorylationofKinases-AProofofConceptStudy.J.Med.Chem.63,2807–2813.


https://doi.org/10.1101/2020.04.04.023713

Intrinsically disordered linkers control tethered kinases ... › content › 10.1101 ›...

Documents

Transcript of Intrinsically disordered linkers control tethered kinases ... › content › 10.1101 ›...