Copper trafficking in eukaryotic systems: current knowledge from experimental and computational efforts

Copper

trafficking

in

eukaryotic

systems:

current

knowledge

from

experimental

and

computational

efforts

Alessandra

Magistrato

1

,

Matic

Pavlin

1

,

Zena

Qasem

2

and

Sharon

Ruthstein

2

Copperplaysavitalroleinfundamentalcellularfunctions,and

itsconcentrationinthecellmustbetightlyregulated,as

dysfunctionofcopperhomeostasisislinkedtosevere

neurologicaldiseasesandcancer.Thisreviewprovidesa

compendiumofcurrentknowledgeregardingthemechanism

ofcoppertransferfromthebloodsystemtotheGolgi

apparatus;thismechanisminvolvesthecoppertransporter

hCtr1,themetallochaperoneAtox1,andtheATPasesATP7A/

B.Wediscusskeyinsightsregardingthestructuraland

functionalpropertiesofthehCtr1-Atox1-ATP7Bcycle,

obtainedfromdiversestudiesrelyingondistinctyet

complementarybiophysical,biochemical,andcomputational

methods.Wefurtheraddressthemechanisticaspectsofthe

cyclethatcontinuetoremainelusive.Theseknowledgegaps

mustbefilledinordertobeabletoharnessourunderstanding

ofcoppertransfertodeveloptherapeuticapproacheswiththe

capacitytomodulatecoppermetabolism.

Addresses

1_{NationalResearchCouncilofItaly-IOMc/oInternationalSchoolfor}

AdvancedStudies(SISSA),viaBonomea165,34135,Trieste,Italy

2

TheChemistryDepartment,FacultyofExactSciences,Bar-Ilan University,529002,Israel

Correspondingauthors:

Magistrato,Alessandra(alessandra.magistrato@sissa.it), Ruthstein,Sharon(Sharon.ruthstein@biu.ac.il)

CurrentOpinioninStructuralBiology2019,58:26–33

ThisreviewcomesfromathemedissueonBiophysicaland com-putationalmethods

EditedbyAlanRLoweandLauraSItzhaki

ForacompleteoverviewseetheIssueandtheEditorial Availableonline6thJune2019

https://doi.org/10.1016/j.sbi.2019.05.002

0959-440X/ã2019TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBYlicense(http://creativecommons. org/licenses/by/4.0/).

Introduction

Copper,likeothermetals,hasapivotalroleinfundamental processesofcellfunction.Ittakespartincellular respira-tion,ironoxidation,pigmentformation,neurotransmitter biosynthesis,antioxidant defense,and connective tissue formation.Yet,whenpresentatexcessiveconcentrations,it canendangerthecell’ssurvival,bycausingde-regulated oxidationofproteins,lipids,andothercellularcomponents, ultimatelyleading toinjury.Moreover,freeCuions can

produceradicaloxygenspecies(ROS),whichcanleadto cytotoxic interactions with cell membranes [1–2,3,4]. Insufficientconcentrationsofcopper,inturn,canleadto metabolic abnormalities, as copper-dependent proteins driveironabsorption,andacopperdeficiencycantherefore lead to iron deficiency. Thus, intracellular pathways of coppermetabolismhaveevolvedtoensuretheappropriate amountofCuforcellsurvival.

Broadly,copper followsthefollowingtrajectorythrough the human body: First, it accumulates in the blood throughdiet.Once it has been ingested,it is taken up from the blood by the copper transporter hCtr1. The copperisthenreducedfromitsoxidizedform,Cu(II),to theCu(I)form;themechanismofreductionisnotfully known,as elaboratedin what follows.Then, the trans-portertranslocatestheCu(I)intothecell.Next,specific Cu(I) chaperones deliver the metal to the appropriate cellularpathways(Figure1)[5–8].Onesuchchaperoneis Atox1,whichtransfersCu(I)toitstransportingATPases intheGolgiapparatus.TheseATPasesinclude ATP7A andATP7B,whichplayabiosyntheticrole,deliveringCu (I)to thesecretory pathway for metalation of cuproen-zymes, and a homeostatic role, exporting excess Cu(I) fromthecell.AdditionalchaperonesincludeCCS,which is required for Cu(I) incorporation into cytoplasmic Cu/Znsuperoxidedismutase,andCox17,whichdelivers Cu(I)to mitochondrialcytochromecoxidase.

In what follows, we provide an overview of current knowledge regarding the hCtr1-Atox1-ATP7B cycle. The hCtr1-Atox1-ATP7A/B cycle is associated with Menkes’ disease and with Wilson’s disease, in which mutations in ATP7A/Bdisrupt the homeostatic copper balance, resultingin Cu deficiency or overload, respec-tively.Besidethese raregeneticdiseases, thecyclehas beenimplicatedinAlzheimer‘sandParkinson‘sdiseases, and in anti-cancer drug resistance [1,2,9–12].Here, we summarizeatomic-leveltypeofinformationonkey resi-duesthataresignificantforfunction,and structuraland kineticinsightsonthecoppertransfermechanismalong its delivery path from the blood system to the Golgi apparatusand/oritsegressfromthecell.

Experimental

and

computational

approaches

used

to

study

copper

trafficking

This review compiles information obtained through diverseexperimentalandcomputationalmethodologies.

These latter include X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, which were usedto solve the3D structuresof thechaperoneAtox1 [13]andthemetal-bindingdomains(MBDs)ofATP7A/

B[14–16].SinglemoleculeFRET(sm-FRET)wasused

toobtainkineticinformationonCu(I)transferfromAtox1 toATP7B[17].Electronparamagneticresonance(EPR) spectroscopy has been used to characterize the various conformationalstatesofAtox1insolutionwhile interact-ing withitspartnerproteins[18].Moreover, ultraviolet-visible spectroscopy (UV-VIS) experiments and cell experimentswereperformedto targetessentialresidues for Cucoordination andfunction.Computational meth-ods suchas moleculardynamics(MD)simulations, rely-ingonforcefields,havebeenappliedtoexplorethefree energylandscapeofproteinmonomersanddimers medi-atingCutransport[19],whereasmixedquantumclassical simulations (QM/MM), ableto overcome limitationsof predefinedforcefields,havebeenemployedtoproperly characterizemetalCu(I)coordinationtotheseproteins,as wellasitsreaction/transfer mechanism[20].

Copper

uptake

by

the

transporter

hCtr1

In1997,ZhouandGitschierbecamethefirsttoidentifya human gene for copper uptake, hCTR1. They showed that each hCtr1 polypeptide contains 190 amino acids [21].TenyearslaterUngeretal.[22,23]reporteda three-dimensional,6-A˚ -resolutionstructureofhCtr1using cryo-genicelectronmicroscopy;theyshowedthattheprotein isatrimercontaining:(1)60aminoacidsinthe extracel-lularN-terminaldomain;(2)threetransmembrane(TM) helices (TM1, 2, and 3); (3) an intracellular loop of 46 amino acids, connecting TM1 and TM2; and (4) a shortintracellularC-terminaldomainwith15aminoacids

(Figure 2).

TheextracellulardomainofhCtr1ischaracterizedbyseveral motifs:glycosylationsites,histidine(His)-richsites,and methionine (Met) motifs. There are two glycosylation sites in hCtr1: N15 and T27. N15 is not required for function[24].Conversely,whenO-glycosylationatT27 isprevented,thefirst30aminoacidsofhCtr1arecleaved [25].

TheHis-richsitesintheextracellulardomainofhCtr1— comprising two motifs, 1MDHxHH and 22HHH—are suggested to serve as Cu(II)binding sites. Thisidea is supported by UV-VIS experiments showing that the extracellular part of hCtr1 can bind two Cu(II) ions [26]. EPR spectroscopy experiments suggest that the processbywhichCu(II)istransferredfromthebloodto hCtr1 involves the blood carrier protein human serum albumin (HSA), as reflected in evidence of close inter-actions between HSA and the N-terminal domain of hCtr1 [27].

The Met-rich motifs in the extracellular domain—two segments, 7MxMxxM and 41MMMxM—are shared by manyproteinsinvolvedin Cu(I)-metabolism;these seg-ments bind Cu(I) with mM affinity [28], and they are essential for hCtr1’s recruitment of Cu(I), produced throughthe reductionof Cu(II)[29,30].Duet al. used UV-VIS titrations to show that three Cu(I) ions can coordinate to the extracellular part of hCtr1 [26]. A combination of EPR, NMR, and UV-VIS experiments performedonthefirst14residuesofhCtr1indicatedthat H5andH6formthefirstCu(II)bindingsite,whereasM7, M9andM12constitutethefirstCu(I)bindingsite[31].

Notably,themechanismoftheCu(II)-to-Cu(I)reduction process remains unclear. It has been suggested that copper and ironmetabolismare intimately linked,with the two metals mutually participating in each other’s oxidation/reduction reactions. This proposition is sup-ported by observations that in the yeast Saccharomyces cerevisiae, both Cu(II) and Fe(III) are reduced in the plasma membrane by Fre1 or Fre2 [32,33], and both Cu(I)and Fe(II)are oxidized(to Cu(II)andto Fe(III), respectively) byFet3metalloxidase[34,35].

The TM domain of hCtr1 is characterized by conserved

150

MxxxMand167GxxxGmotifs.DeFeoetal.identified M150 and M154 in TM2 as Cu(I)-binding residues, whereas G167 and G171, both located in TM3, were proposed to mediate a tight interface between TM1 and TM3 [23]. Schushan et al. constructed a Ca-trace model of theTM domain of hCtr1, which agreed well withtheexperimentalstructure[36].UsingaGaussian network modelandan anisotropicnetwork model,they identifiedpossiblefunctionalmotionsoftheTMhelices. On the basis of these motions, the authors proposed a transport mechanism in which the Cu(I) ions are transferred one at a time, and M154 (which points to

Figure1 Ctr1 Atox1 Cox17 CCS SOD Cu(I) Cu(I) Cu(I) Cu(I) Cu(II) ATP7B

Current Opinion in Structural Biology

theextracellularpartofthemembrane)alongwithH139 and E84 (conserved residues) control the transporter’s motionasafunctionofmetalionbindingandapHshift. This model is supported by biochemical experiments showingthatH139andE84areindeedimportant func-tional residues [37,38]. Tsigelny et al. used all-atom simulations to developamodelfor the fullstructure of hCtr1[39];usingaCatraceapproach,theysuggestedthat M43,M45,M150, andM154, andthe188HCHmotif in theC-terminaldomain areimportant residues forCu(I) binding.

The intracellular domain of hCtr1 contains two parts: an intracellular loop between TM1 and TM2, and a short C-terminal tail. The last 15 residues, ending with a

188

HCH motif, are essential for Cu(I) transport. Kaplan andco-workers used 64Cu cellexperiments to showthat

C189isnot essentialfor Cu(I)uptake[24], butthat188HCH residues arevitalfor transferandregulationpurposes [37].In theintracellulardomainC189wasfoundtobecriticalforCu (I) bindingand transfer to Atox1[37,40].NMR experi-ments revealed that Cu(I) binds to 188HCH with high affinity (KD of 10 14 M) [41]. Asa result, Cu(I) can be

releasedtoitstargetbyprotein–proteininteraction,while beingunabletofreelydissociatefromhCtr1.Arecentstudy that used EPR along withcircular dichroism (CD) and NMR [42]showedthattheintracellularloopcanformasecond low-affinityCu(I)-bindingsite(KD 1–10mM)involving

theresiduesM106,M117,andH120.

Distribution

of

Cu(I)

by

the

metallochaperone

Atox1

Atox1, alsocalled Hah1,is asoluble protein(68 amino acids),displayingababbabfold[13].Itcoordinatesone

Figure2

Cu(II) binding residues

Cu(I) binding residues Glycosylation residues

Other functional residues

COOH H₂N

Current Opinion in Structural Biology

Cu(I) ion with the cysteine residues of a conserved 10MxCxxC motif (Figure 3). Cu(I)’s binding affinity towardAtox1isevenhigherthanthatfortheC-terminal domain of hCtr1(KD=10 17.4M),enabling Cu(I)to be

transferredfromhCtr1 tothemetallochaperone[43].

DataobtainedfromthecrystalstructureofAtox1,aswell as from NMR experiments and QM/MM simulations, point to additional residues, besides the conserved Cys-based motif that are important to Atox1 function. Specifically, K60was foundtobeimportantfor neutral-izingthenegative chargein theCu(I)bindingsite, and T11 was shown to contribute to the Atox1’s flexibility

[44–46].Additionally,M10,completelyconservedacross

different organisms, is buried in thehydrophobic core, contributingtoAtox1’sstability[47].Far-UVCDtitration experiments in the pH range 6–11 showed that Cu(I) affinityfor Atox1decreaseswiththepHlevel,owingto protonation of the cysteine residuesin the bindingsite [48]. Considering that hypoxic malignant cells are

characterized by low pH, this finding might point to a mechanism bywhich cancerinterfereswith Cu-homeo-stasis [49,50].

Notably,sm-FRETexperimentshavedemonstratedthat Atox1canassumetwoconformationalstates,bothableto interact with the target protein ATP7B [17]. EPR measurementstogetherwithcomputationshaveresolved thesetwo conformationalstates[18,40,42],further sug-gestingthatAtox1canadoptconformationsspecifictoits targetprotein. EPRexperimentshavealso revealedthe importance of 188HCH in stabilizing thecomplex com-prisingtheC-terminaltailofhCtr1andAtox1[40],and haveconfirmedthatAtox1interactswiththeintracellular domainofhCtr1(boththeC-terminaltailandthe intra-cellularloop)asahomodimer.TheAtox1homodimerhas been solved by X-ray studies showing that the Atox1 dimer is stabilized bymetal-mediated interactions,and by complementarity in the electrostatic interactions betweenthetwomonomers[44].

Figure3 (a) (b) (c) S S S HS SH SH SH S S S S S S HS S Cu(I) Cu(I) Cu(I) Cu(I) HS

90°

90°

Atox1 MBD4

Current Opinion in Structural Biology

(a)ProposedmechanismforCu(I)transferfromAtox1toMBD4.(b)RepresentativestructureobtainedbyMDfortheinteractionbetween holo-Atox1andMBD4.(c)Thestructureandelectrostaticpotentialsurfaceofholo-Atox1monomerandMBD4.

Cu(I)

uptake

by

the

metal-binding

domains

of

ATP7B

ATP7B is made of eight TM segments and two large cytosolicdomains:(i)theN-terminalCu-bindingdomain, and(ii)thecatalyticATPhydrolyzingdomain[51].Over 300mutationsinATP7Bhavebeenidentifiedthataffect its function, indicating that almost one-third of the encodedresiduesarestrictlyrequiredforproperATP7B function.Theseobservationssuggestthat,beyond iden-tifyingpoint mutationsthat affectCu-homeostasis, itis necessarytoobtainadetailedmechanisticpictureofCu (I)transfer inorderto unravelthemolecularbasisofits metabolism.

Atox1interacts withtheN-terminal domainof ATP7B, whichcontainssixMBDsconnectedbylinkers.Similarto

theAtox1metallochaperone,eachMBDhasa ferredoxin-like fold with a compact babbab structure and a con-served metal-binding motif MxCxxC, located in the solvent-exposed b1-a1 loop. On the basisof the struc-tures of Atox1 and of an MBD of ATP7B, Wernimont etal.proposedamechanismforCu(I)transferfromAtox1

totheMBD(Figure3).InthismodelAtox1transfersCu

(I)toasingleMBDthroughconsecutiveligandexchange reactions[44].Subsequentstudiesusedbiophysicaland computational methods to characterize the interactions betweenAtox1andthesixMBDsofATP7A/B,focusing mostly on MBD4. NMR studies showed that the six MBD units (MBD1–6) can be differentiated into two groups,comprisingMBD1–3,andMBD5–6,withMBD4 servingas alinkerbetweenthem [14,44,52].The struc-turesofMBD3andMBD4werealsosolvedusingNMR

Figure4 hCtr1 N-terminal domain hCtr1 C-terminal domain hCtr1 Atox1 dimer Atox1 monomer MBD (ATP7B) ATP7B COOH MBD6 MBD2 _MBD3 MBD4 MBD5 MBD1 H2N ADP ATP hCtr1 intracellular loop

Current Opinion in Structural Biology

hCtr1-Atox1-ATP7BCu(I)transfermodel.Atox1playsacriticalroleinmediatingCu(I)transfermechanism,cyclingbetweenadimertomonomer stateandadjustingaspecificconformationbasedonitstargetprotein.

[15,53].OfalltheMBDs,Atox1 interactsmoststrongly with MBD1-MBD4, whereas it does not interact with MBD5 or with MBD6 [14,16,54,55]. Cu(I) binding to regulatory MBD1–4 stimulates its transport byATP7B and presumably facilitates the trafficking of the trans-porter by exposing sites for further modifications and protein–protein interactions [15,56]. However, NMR studiesandMDsimulationsrevealedanexclusive inter-action betweenAtox1 andMBD4, andnotwith MBD3 [19,54]. Whereas, smFRET experiments identified a dynamic situation in which, because of its structural flexibility, Atox1 can coordinateeither MBD3, MBD4, or the two domains simultaneously, MBD3–4 [17]. A recent studyby ourgroup, relying ona combinationof EPRandMDsimulations,revealedthatCu(I)extrusion is mostlikelymediatedbyAtox1bindingto MBD4via theformationoftransientinteractionsmediatedby elec-trostaticcomplementarityofthetwosurfaces.Regarding thechemicalmechanism,Cu(I)appearstobetransferred byligandexchangefromC12/C15ofAtox1toC370/C373 of MBD4 via the formation of several intermediates displaying a three-coordinated Cu(I) site (Figure 3). QM/MM simulations suggest that K60 of Atox1 may actively modulate the Cu(I) exchange [54]. All these experiments indicate that Atox1 interacts with MBDs inamonomericstate[57,58],incontrasttoitsinteraction with hCtr1, as a dimer (Figure 3). Figure 4 shows a mechanistic picture of Cu(I) transfer from hCtr1 to MBDof ATP7Bbasedonthecurrentknowledge.

Conclusions

Inrecentyears,significantprogresshasbeenmadetoward elucidatingthemechanismsunderlyingintracellular cop-perregulation.Multipledistinctstudieshaveconsistently revealed that the methionine segments and cysteine-based motifs present in thevarious components of the hCtr1-Atox1-ATP7A/B cycleare criticalfor Cu(I) bind-ing. Studies investigating the effect of pH on Cu(I) affinitytoproteinshaveprovidedcriticalinsights regard-ingthepossiblemechanismsofcysteineligandexchange reactionsunderlyingmetaltransport.Discoveries regard-ingtheconformationalflexibilityofAtox1,togetherwith itscapacitytofunctioneitherinadimericormonomeric form,dependingonitsproteinpartner,indicatehowthe metallochaperoneelegantlymediatesCu(I)transferfrom hCtr1totheGolgiapparatusand/ortoitsexcretionroute.

Inspiteoftheseachievements,manygapsremaininour understanding of this important metabolic pathway. In particular, knowledge of the mechanisms underlying hCtr1andATP7A/Bfunctionremainsfragmentary,owing to the challenges involved in expressing and purifying theseproteinsforbiophysicalresearch,andinresolvinga structuralmodelatatomic-levelresolution.Itisnecessary to overcome these obstacles, using a combination of biophysical,biochemical,andcomputationalapproaches, inordertoidentifyallCu(I)bindingsites,andtoobtaina

comprehensiveunderstandingoftheCu(I)transfer mech-anism. As discussed above, current experimental and computational data suggest that each MBD in ATP7B hasaspecific role;these observationsshould befurther analyzedandconfirmedonthefull-lengthATP7B.

AmorecompleteunderstandingofhCtr1-Atox1-ATP7A/ Bcyclemaycontributetowardthedevelopmentof treat-ment for diseases associated with Cu-metabolism dys-function,includingMenkes’andWilson’sdiseases. Cur-renttreatmentfortheseconditionsreliesonCu-chelators, whichhaveledtoanincreasedlifespaninsomepatients, yet are not uniformly effective. Moreover, although numerous studies have identified connections between copper regulation and neurological diseases such as Parkinson’s, Alzheimer’sand priondiseases, the under-lyingmechanismsoftheseconnectionsremainselusive. Similarly, the role of Cu-transport and homeostasis in cancer,andinresistancetocommonlyusedmetal-based anticancerdrugs, remainsunresolved[9,10].

Herein,wehavesoughttodepictcurrentunderstanding oftheCu-transfermechanismattheatomiclevel, empha-sizing the delicate balance of transient protein–protein interactions underlying metal transfer, reactivity, and homeostasis. Itremainsadaunting challengetoharness this knowledge for future innovative therapeutic approaches aimingatcounteractingthemany pathologi-calstatesassociatedwithCu-dis-homeostasis.

Conflict

of

interest

statement

Nothingdeclared.

Acknowledgements

ThisworkwassupportedbytheIsraelandItalianMinistryofSciencegrant giventoSRandAMandbytheERC-STGgrantno.754365giventoSR. AMthankstheItalianAssociationforCancerResearch(MFAGGrantno. 17134).

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