MetallothioneinsMetallothioneins

An overviewAn overviewAn overviewAn overview

Òscar PalaciosÒscar PalaciosDept QuímicaDept QuímicaDept. QuímicaDept. QuímicaUniversitat Autònoma de BarcelonaUniversitat Autònoma de Barcelona

MetallothioneinsMetallothioneins ((MTsMTs))MetallothioneinsMetallothioneins ((MTsMTs))

Main Goal:

Metallothioneins are not “normal” or “ ti l” t i / t ll t i“conventional” proteins/metalloproteins: they deserve special attention and must be t t d f lltreated carefully

ProteinsProteins//MetalloproteinsMetalloproteinsProteinsProteins//MetalloproteinsMetalloproteinsProteinsProteins

Big “size”: 10-150 kDa 3D structure determined by

sequencesequence Presence of secondary

motivesC d t f S S Cys used to form S-S bridges

All kind of amino acids Metalloproteins

3D structure determined by ( )(related with function) sequence (most)

Cys, His, Asp, Glu, Met used to bind metal ions

Variety of functions (related with metals sometimes)

ProteinsProteins//MetalloproteinsMetalloproteinsProteinsProteins//MetalloproteinsMetalloproteins

Hemoglobin (Fe)575 aa, 64 kDa

YiiP zinc transporter protein in Escherichia coli (Lu & Fu, 2007,2009)

300 aa, 33 kDa

MetallothioneinsMetallothioneins ((MTsMTs))MetallothioneinsMetallothioneins ((MTsMTs))Discovered in 1957 (Margoshes andDiscovered in 1957 (Margoshes and

Vallee)Horse kidney protein Horse kidney protein

High metal content (Zn2+ and Cd2+) High Cys content in the primary sequence High Cys content in the primary sequence Absence of aromatic amino acids

MDPNCSCASDGSCSCAGACKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEASDKCSCCA

61 aa, 20 Cys, 7 M2+ content

MetallothioneinsMetallothioneins ((MTsMTs))MetallothioneinsMetallothioneins ((MTsMTs))Subsequently discovered in other organismsSubsequently discovered in other organisms Two very similar MT isoforms in mammals (MT1 and MT2):

60-62 aa, 20 CysC bi di i i (C 1) 55 12 C Cu-binding protein in yeast (Cup1): 55 aa, 12 Cys

Human MT1 MDPNCSCAAGVSCTCAGSCKCKECKCTSCKKSCCSCCPVGCSKCAQGCVCK

S. cerevisiae Cup1MQNEGHECQCQCGSCKNNEQCQKSCSCPTGCNSDDKCPCGNKSEETKKSCCSGK

GASEKCSCCD

Higher organismsAb t 60

Lower organisms- Short aa sequence- About 60 aa

- Binding M(II)- Formation of M7-MT clusters(M = Zn Cd)

Short aa sequence- Cu(I)-binding- Formation of only-Cuclusters(M = Zn, Cd)

- Crub, sea urchin, fish,... y ,- Yeast, fungui, nematode, cyanobacteria,...

MetallothioneinsMetallothioneins ((MTsMTs))MetallothioneinsMetallothioneins ((MTsMTs))Oth fi di fi i th t t fOther findings confirming the two types of

MTsMTs 3D structural determination

Zn7-MT1Cd MT1

Cu7-Cup1 Ag7-Cup1

Cd7-MT1(mammalian)

(yeast)

MammalianMammalian MTMTCd5Zn2-MT2 (X-ray difraction)

MammalianMammalian MTMTCd5Zn2 MT2 (X ray difraction)

Domain (C-t)

(M4S11)

Domain (N-t)

(M S )

Robbins et al., J. Mol. Biol., (1991), 221, 1269

(M3S9)

MetallothioneinsMetallothioneins ((MTsMTs))MetallothioneinsMetallothioneins ((MTsMTs))ReactivityReactivity Binding of different metal ions (in vivo and in vitro):

Zn(II), Cd(II), Hg(II), Cu(I), Ag(I), Au(I), Bi(III), As(III), Co(II), Fe(II), Pb(II) Pt(II) d T (IV)Pb(II), Pt(II) and Tc(IV)

Exchange of metal ions following the Irving-Williams series:series:

Fe(II)≈Zn(II)≈Co(II)<Pb(II)<Cd(II)<Cu(I)<Au(I)≈Ag(I)<Hg(II)<Bi(III) Metal transfer between proteins and other

biomoleculesbiomolecules Formation of S-S bridges when interaction with

oxidative species (release of metal ions)oxidative species (release of metal ions) Interaction with radicals ...

MetallothioneinsMetallothioneins ((MTsMTs))MetallothioneinsMetallothioneins ((MTsMTs))Proposed functions (related withProposed functions (related with

reactivity)H l d ifi i Heavy metal detoxification

Homeostasis of essential ions (Zn2+ and Cu+) Metal chaperone role Redox activityy Protection against free radicals Role in the energy metabolism Role in the energy metabolism

ClassificationClassification ofof MTsMTsClassificationClassification of of MTsMTsNon-conventional criteriaNon-conventional criteria First attempt: similarity of sequences Class 1: MTs similar to mamalian MT1 Class 1: MTs similar to mamalian MT1

and MT2 Class 2: MTs different of class 1 proteins Class 2: MTs different of class 1 proteins Class 3: Phytochelatins and cadistins

Second attempt: strict analysis of aa Second attempt: strict analysis of aasequences

Third attempt: gradation between Zn- and p gCu-thioneins

Characterization at the early stagesCharacterization at the early stagesCharacterization at the early stagesCharacterization at the early stages

StructuralSpectroscopical

UV-vis absorptionNMRX-ray

Circular dichroism(CD) and MCD

X ray

FluorescenceELISA, RIAAnalytical

ICP-AES

SDS-PAGE, HPLC

ICP-AESAAS Metallothineins, M. J. Stillman, C. F. Shaw III and K.

T. Suzuki (Eds.), 1992, VCH Publishers

Diverse MTs discovered and Diverse MTs discovered and studiedstudied

> 200 MT sequences~ 110 organisms

UniProtKB/(Swiss-Pro and TrEMBL) databases

Diverse MTs discovered and Diverse MTs discovered and studiedstudied

H MT1Human MT1MDPNCSCAAGVSCTCAGSCKCKECKCTSCKKSCCSCCPVGCSKCAQGCVCKGASEKCSCCD

C.elegans MT1MACKCDCKNKQCKCGDKCECSGDKCCEKYCCEEASEKKCCPAGCKGDCKCANCHCAEQKQCGDKTHQHQGTAAAH

MPCPCGSGCKCASQATKGSCNCGSDCKCGGDKKSACGCSE

D.melanogaster MTN

QsMT, plant MTMSCCGGNCGCGTGCKCGSGCGGCKMFPDISSEKTTTETLIVGVAPQKTHFEGSEMGVGAENGCKCGSNCTCDPCNCK

TpyMT1, Tetrahymena MT

MGVGAENGCKCGSNCTCDPCNCK

MDKVNNNCCCGENAKPCCTDPNSGCCCVSETNNCCKSDKKECCTGTGEGCKWTGCKCCQPAKSGCCCGDKAKACCTDPNSGCCCSSKTNKCCDSTNKTECKTCECCK

CharacterizationCharacterization actuallyactuallyCharacterizationCharacterization actuallyactuallyRecombinant

UV-vis absorption Fluorescence

Mass spectrometryStructural

Spectroscopical

synthesis

Multinuclear NMR

Mass spectrometry

UV-vis absorptionX-ray

Circular dichroism(CD) and MCD

FluorescenceAnalytical

ICP-AESHPLC Problems with

conventionaltechniquesICP AES

ICP-MStechniques

MammalianMammalian MTsMTsMammalianMammalian MTsMTs Four isoforms: MT1 MT2 MT3 MT4 Four isoforms: MT1, MT2, MT3, MT4 MT1 and MT2: ubiquitous MT3: brain (also known as GIF) MT3: brain (also known as GIF) MT4: skin

Similar amino acid sequencesq Different reactivity (function?)

MT1 MDPN-CSCSTGGSCTCTSSCACKNCKCTSCKKSCCSCCPVGCSKCAQGCVCKGA------ADKCTCCA 61 MT2 MDPN-CSCASDGSCSCAGACKCKQCKCTSCKKSCCSCCPVGCAKCSQGCICKEA------SDKCSCCA 61 4 62MT4 MDPGECTCMSGGICICGDNCKCTTCSCKTCRKSCCPCCPPGCAKCARGCICKGG------SDKCSCCP 62

MT3 MDPETCPCPTGGSCTCSDKCKCKGCKCTNCKKSCCSCCPAGCEKCAKDCVCKGEEGAKAEAEKCSCCQ 68

PlantPlant MTsMTsPlant Plant MTsMTs The less known members of the MT superfamily 1st plant MT isolated 25 years after MTs in horse 1 plant MT isolated 25 years after MTs in horse Divided into 4 subfamilies, depending on the number of

Cys residues and their distribution in the polypeptidicsequence

10 20 30 40 50 60 70 80 90 ....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|..

MSCK-----CGSNCSCGSSCNCNSCDVEKS----TTTTIIVDGVAPKMTFAEGSETSTVAESGNGCSCGSSCKCNPCNC- 70 MSSCG----CGSSCNCGSNCGCNKYSFDLSYVEKTTTETLVLGVGPVKAQLEGAEMGVASENG-GCNCGSSCTCDPCNCK 75 MSCSSGKCNCGSSCSCGSSCNCNSCNVEMST---TTTTIIVDGVAPRMTFAEETE---VAESGNACKCGSSCKCDPCNC- 73

MS--CCNGKCGCGSSCSCGSGCNGCGMYPDV---EVSSTTVMIVDGVAPKQMFAEGSEGSFVAEGGNCNCKCGDNCKCGNNCSC- 79 MS--CCGGNCGCGSGCKCGNGCGGCKMYPDLGFSGETTTTETFVLGVAPAMKNQYEASGESNNAENDA-CKCGSDCKC-DPCTCK 81

1

2 MS CCGGNCGCGSGCKCGNGCGGCKMYPDLGFSGETTTTETFVLGVAPAMKNQYEASGESNNAENDA CKCGSDCKC DPCTCK 81 MS--CCGGNCGCGSACKCGNGCGGCKMYPDL-SYTESTTTETLVMGVAPVKAQFES-AEMGVPAENDG-CKCGANCTC-NPCTCK 79 MS--CCNGKCGCGSSCSCGSSCKGCGMYPDI---ETSTTATMIVDGVAPKKMYDDGSDASFVSEGGHA-CKCGANCKC-DPCNC- 77 MSSSCCGGNCGCGSGCKCGNGCGGCKMYPEM-VSGESTNNETLILGVAPTETGIETTGGVATAAENEG-CKCNP-CTC-NPCTCK 81

MS-SCTNCDCFDKANCTKKGFRYDVTVVETETSTEETNVMEVPAAENNG--NCKCGANCSCTNCTCGH- 65 MSSNCGSCDCADKTQCVKKGTSYTFDIVETQESYKEAMIMDVGAEENNANCKCKCGSSCSCVNCTCCPN 69

2

3 MSSNCGSCDCADKTQCVKKGTSYTFDIVETQESYKEAMIMDVGAEENNANCKCKCGSSCSCVNCTCCPN 69 MSNTCGNCDCADKTSCTK-GNSYGV-IVETEKSYIETVVMDVPAAEHDG--KCKCGTNCTCTDCTCGH- 64

---MRG-------VICDERCGCPSPCPGGVSCRCKSGRMESGGGEVEHKKCSCGGHCGCNPCSCSQA-TPSEGTGKAFCKCADGCTCVTCSS 81 MADTSGGDAVRPVVICDNKCGCTVPCTGGSTCRCTSVGMTTGGG--DHVTCSCGEYCGCNPCSCPKT-AAS-GTG---CRCGTDCSCASCRT 85 MADTGKGSS---VAGCNDSCGCPSPCPGGNSCRCRMREASAGDQ--GHMVCPCGEHCGCNPCNCPKTQTQTSAKG---CTCGEGCTCASCAT 84

3

4

Cobbett et al., Annu. Rev. Plant. Biol., 2002, 53, 159‐82

DrosophilaDrosophila MTsMTsDrosophilaDrosophila MTsMTs Five isoforms Five isoforms Similar length and Cys content Cu(I)-binding preferencesCu(I) binding preferences

1 10 20 30 40 MtnA MPCP-CGSGCKCASQATKGSCNCGSDCKC---GGDKKSACGCSE 10 CysMtnB MVCKGCGTNCQCSAQKCGDNCACNKDCQCVCKNGPKDQCCSNK 12 CysMtnC MVCKGCGTNCKCQDTKCGDNCACNQDCKCVCKNGPKDQCCKSK 12 CysMtnD MGCKACGTNCQCSATKCGDNCACSQQCQCSCKNGPKDKCCSTKN 12 CysMtnE MPCKGCGNNCQCSAGKCGGNCAGNSQCQCAAKTGA--KCCQAK 10 CysMtnE MPCKGCGNNCQCSAGKCGGNCAGNSQCQCAAKTGA KCCQAK 10 Cys

Snail MTsSnail MTsSnail MTsSnail MTs Terrestrial snails with metal-specific MT isoformsp

Helix pomatia: HpCuMT and HpCdMT Cornu aspersum: CaCuMT, CaCdMT and CaCdCuMT Similar length and amino acid content among them Similar length and amino acid content among them Perfect Cys homology Different in vivo and in vitro metal specificity

HpCuMT GS—-GRGKNCGGACNSNPCSCGNDCKCGAGCNCDRCSSCHCSNDDCKCGSQCTGSGSCKCGSACGCK HpCdMT GSGKGKGEKCTSACRSEPCQCGSKCQCGEGCTCAACKTCNCTSDGCKCGKECTGPDSCKCGSSCSCK

CaCdMT GSMSGKGKGEKCTAACRNEPCQCGSKCQCGEGCTCAACKTCNCTSDGCKCGKECTGPDSCKCGSSCGCK CaCdCuMT GSMSGKGSA--CAGSCNSNPCSCGDDCKCGAGCSCAQCYSCQCNNDTCKCGSQCSTSGSCKCGS-CGCK CaCuMT GSMSGRGQN—-CGGACNSNPCNCGNDCNCGTGCNCDQCSARHCSNDDCKCGSQCTRSGSCKCGNACGCK

Native: Cu12-HpCuMT & Cd6-HpCdMT

(Gehrig et al., Prot. Science 2000) (Dallinger et al., Nature 1997)

DiversityDiversity of 3Dof 3D structuresstructuresDiversityDiversity of 3D of 3D structuresstructuresHuman Cd7-MT2 domains7

S. elongatus Zn4–SmtA

M. Capdevila et al. / Coordination Chemistry Reviews 256 (2012) 46– 62

DiversityDiversity of 3Dof 3D structuresstructuresDiversityDiversity of 3D of 3D structuresstructures

T. aestivum Zn4-E-Ec-1

Zn(SCys)2(NHis)2 Zn3(SCys)9

S. cerevisiae Cu8-Cup1

M. Capdevila et al. / Coordination Chemistry Reviews 256 (2012) 46– 62

MTs in medicine & biotechnologyMTs in medicine & biotechnologyMTs in medicine & biotechnologyMTs in medicine & biotechnology Involved in many biological processes: Involved in many biological processes:

biomedical applications Protection against Pt-drugs Protection against Pt drugs Tumor prognosis and biomarker Treatment of neurodegenerative diseasesg Help at symptomological level in several

disorders Composition and in vitro reactivity:

biotechnological applications Bioremediation of metal-poluted areas Biosensor to monitor metal

accumulations

MetallothioneinsMetallothioneins ((MTsMTs))MetallothioneinsMetallothioneins ((MTsMTs)) Ubiquitous proteins: animals plants Ubiquitous proteins: animals, plants,

prokaryotes Small metalloproteins (40-100 aa)Small metalloproteins (40 100 aa) High sequence heterogeneity General absence of aromatic amino acids Cysteine-rich (~ 30 %) High metal-chelating capacity (thiol groups)g g p y ( g p ) No 3D structure of the apo form

structuration related with the formation of metal clusters

Biological role: metal homeostasis and detoxification, radical scavenging, …

MetallothioneinsMetallothioneins

An overviewAn overviewAn overviewAn overview

Òscar PalaciosÒscar PalaciosDept QuímicaDept QuímicaDept. QuímicaDept. QuímicaUniversitat Autònoma de BarcelonaUniversitat Autònoma de Barcelona