Structure and evolution Structure and evolution of IDPsof IDPs

Peter Tompa

Institute of EnzymologyHungarian Academy of Sciences

Budapest, Hungary

Why do we want to Why do we want to characterize/predict IDPs?characterize/predict IDPs?

1) Find new ones (460 in 1) Find new ones (460 in DisProt vs. DisProt vs. tens of thousands)tens of thousands)

2) Describe our 2) Describe our proteinprotein

Extend the structure-function Extend the structure-function paradigmparadigm

Why do we want to describe the Why do we want to describe the structure of IDPs in detail?structure of IDPs in detail?

To characterize…

Structure

In the bound state

In the free state

Structural levels

Structure

Local (secondary)

Global (tertiary)

Sequence (primary)

1) Primary structure

Dunker et al. (2001) J. Mol. Graph. Model. 19, 26

Primary structure (sequence) of Primary structure (sequence) of IDPsIDPs

Low-complexity regions in proteins

Wootton (1994) Comp. Chem. 18, 269

Low complexity:Low complexity: Drosophila Drosophila mastermindmastermind

MDAGGLPVFQSASQAAAAAVAQQQQQQQQQQQQQQQQQQQHLNLQLHQQHLQQQQSLGIHLQQQQQLQLQQQQQHNAQAQQQ

QQLQVQQQQQQRQQQQQQQQQHSLYNANLAAAGGIVGGLVPGGNGAGGVALQQVFGGPNGNNNSNNNNNSNNNSININNGNI

SPGDGLPTKRQPILDRLRRRMENYRRRQTDCVPRYEQTFSTVCEQQNHETSALQKRFLESKNKRAAKKTEKKLPETQQQAQT

QMLAGQLQSSVHVQQKILKRPADDVDNGAENYEPPQKLPNNNNNNNNNNNNNNNSSSGVGGGSENLTKFSVEIVQQLEFTTS

AANSQPQQISTNVTVKALTNTSVKSEPGVGGGRGRHQQQQQHQQHQQQQHQQQQHQQHQQHQQQQQHQQQQHQQQQHQQQQQ

QHHHQQQQQQGGGLGGLGNNGRGGGGPGGGGHMATGPGGVGVGMGPNMMSAQQKSALGNLANLVECKREPDHDFPDLGSLAK

DGANGQFPGFPDLLGDDNSENNDTFKDLINNLHDFNPSFLDGFDEKPLLDIKTEDGIKVEPPNAQDLINSLNVKSETGLGHG

FGGFGVGLGLDPQSMKMRPGVGFQNGPNGNANAGNGGPTAGGGGGGNGPGGLMSEHSLAAQTLKQMAEQHQHKSAMGGMGGF

HVPPHGMQQQQPQQQQQAPQQQQQQHGQMMGGPGQGQQQQQQQQPRYNDYGGGFPNDFAMGPNPTQQQQQHLPPQFHQKAPG

GGPGMNVQQNFLDIKQELFYSSPNDFDLKHLQQQQAMQQQQQQQQQQQQQQQHHAQQQQQHPNGPNMGVPMGGAGNFAKQQQ

QQVPTPQQQQQQQLQQQQQQYSPFSNQNANANFLNCPPRGGPQGNQAPGNMPQQQQQQPQQQQQPPRGPQSNPNAVPGGNAA

NATQQQQQQQQQQQQQQQQQQQQQQQATTTTLQMKQTQQLHISQQGGGSHGIQVSAGQHLHLSSDMKSNVSVAAQQGVFFSQ

QQAAQQQQQQQQQPGNAGPNPQQQQQQPHGGNAGANGGGPNGPQQQQPNQNMNNSNVPSDGFSLSQSQSMNFTQQQQQQAAA

AAAAAAAAQQQQAAAAQQQQQQVPPNMRQRQTQAQAAAAAAAAAAAQAQAAANANGGPGGNVPLMQQQQQTPGGVPVGAGSG

NASVGVPVSAGGPNNGAMNQLGGPMGGMPGMQMGGPGGVPINPMQMNPNGGAPNAQMMMGGNGGGPVPAASQAKFLQQQQIM

RAQAMQHQQQVQQHMAGARPPPPEYNATKAQLMQAQMMQQTVGGGGGGGVGVGVGVGGGVGGGGGAGRFPNSAAQAAAMRRM

TQQPIPPSGPMMRPQHAAMYMQQHGGAGGGPRGGMGGPYGGGGVGGAGGPMGGGGGGQQQQQRPPNVQVTPDGMPMGSQQEW

RHMMMTQQQQQMGFGPGGPMRQGPGGFNGGNFMPNGAPNAPGNGPNGGGGGGMMPGPNGPQMQLTPAQMQQQHMRQQQQQQH

MGPGGGGGGGGGNMQMQQLLQQQQNAAAGGGGGMMATQMQMTSIHMSQTQQQQQLTMQQQQFVQSTSTTTTHQQQQQLQLQM

QSQSGGPGGNGPSNNNGANQAGGVGVGVGVGVGVGVVGSSATIASASSISQTINSVVANSNDLCLEFLDNLPDGNFSTQDLI

NSLDNDNFNIQDILQ

Drosophila Drosophila mastermindmastermind

2) Secondary structure

Structure in the free state (3 examples)

CREB-KID - CBP-KIX binding and NMR

Radhakrishnan et al. (1998) FEBS Lett. 430, 317

FlgM: evidence for disorder in vivo

Plaxco and Gross (1997) Nature, 386, 657

Sorenson (2004) Mol. Cell 14, 127

FlgM - sigma 28 binding and NMR

p27 p27 – CycA/Cdk2 binding– CycA/Cdk2 binding (NMR, MD) (NMR, MD)

Sivakolundu et al. (2005) JMB 353, 1118

Wikipedia

SH3-PPII

And a fourth: polyproline II helixAnd a fourth: polyproline II helix

PPII helix conformation is common in IDPs

Raman optical activity (ROA)

Syme et al. (2002) EJB 269, 148

PPII

Dominates in : -casein -synuclein tau wheat gluten

2) Secondary structure

Structure in the bound state

Complexes of IDPs in PDB

p27Kip1 IA3

FnBP

Tcf3

CycA

Cdk2

fibronectin

-catenin

Asp prot.

IUP SP code Length Partner Method CREB P16220 28 CBP KIX NMR

DFF 45 O00273 89 DFF 40 NMR

E-cadherin P09803 57 -catenin X-ray

FCP1 AAC64549.1* 21 TFIIF/RAP74 NMR

FnBPA Q53971 24 Fibronectin NMR

IA3 P01094 29 Proteinase A X-ray

Killer toxin P19972 77 Killer toxin chain X-ray

Bob-1 P10636 13 Pin1 WW NMR

MAP tau P25912 86 DNA X-ray

MAX Q16633 22 Oct-1 POU/DNA X-ray

p27Kip1 P46527 69 CycA-Cdk2 X-ray

p53 P04637 11 MDM 2 X-ray

Phe-tRNA synthetase P27001 79 Phe-tRNA synthetase + tRNA X-ray

PKI P04541 20 PKA X-ray

RB3 Q9H169 91 tubulin X-ray

RNA pol II P04050 17 mRNA capping enzyme X-ray

SNAP 25 P13795-2 77 neuronal fusion complex X-ray

SV 40 virus coat P03087 66 assembled coat X-ray

TAFII230 P51123 67 TBP NMR

TBS virus coat P11795 34 assembled coat X-ray

Tcf3 CAA67686* 41 -catenin X-ray

Tcf4 Q9NQB0 24 -catenin X-ray

Troponin I P19429 17 Troponin C NMR

Vitamin D3R P11473 89 DNA X-ray

0 20 40 60 80 1000

20

40

60

80

100

Hélixfe

hér

jék

%-a

másodlagos szerkezet %-a

globularIDP

0 20 40 60 80 1000

20

40

60

80

100Extended

feh

érjé

k %

-a

másodlagos szerkezet %-a

0 20 40 60 80 1000

20

40

60

80

100

Turn

feh

érjé

k %

-a

másodlagos szerkezet %-a

0 20 40 60 80 1000

20

40

60

80

100

Coil

feh

érjé

k %

-a

másodlagos szerkezet %-a

Secondary structural elements

31.3 %44.8 %

21.9 %10.9 %

Helix

Comparison of free and bound states:

what does it tell us ?

Local secondary structural elements in IDPs:

molecular recognition

1) disorder pattern molecular recognition element

MoRE, MoRF2) consensus sequence:

linear motifLM, ELM, SLiM

3) local predictable structurepreformed structural element

PSE

1) Disorder pattern: MoRE in tumor suppressor p53

Uversky et al. (2005) J. Mol. Recogn. 18, 343

2) Consensus sequences: ELMs2) Consensus sequences: ELMs

ELMs and local disorderELMs and local disorder

Fuxreiter et al (2006) Bioinformatics, 23, 950

3) Predictability of structure: 3) Predictability of structure: preformed structural elements, preformed structural elements,

PSEsPSEs

p27Kip1 IA3

FnBP

Tcf3

CycA

Cdk2

fibronectin-catenin

Asp prot.

IUP SP code Length Partner Method CREB P16220 28 CBP KIX NMR

DFF 45 O00273 89 DFF 40 NMR

E-cadherin P09803 57 -catenin X-ray

FCP1 AAC64549.1* 21 TFIIF/RAP74 NMR

FnBPA Q53971 24 Fibronectin NMR

IA3 P01094 29 Proteinase A X-ray

Killer toxin P19972 77 Killer toxin chain X-ray

Bob-1 P10636 13 Pin1 WW NMR

MAP tau P25912 86 DNA X-ray

MAX Q16633 22 Oct-1 POU/DNA X-ray

p27Kip1 P46527 69 CycA-Cdk2 X-ray

p53 P04637 11 MDM 2 X-ray

Phe-tRNA synthetase P27001 79 Phe-tRNA synthetase + tRNA X-ray

PKI P04541 20 PKA X-ray

RB3 Q9H169 91 tubulin X-ray

RNA pol II P04050 17 mRNA capping enzyme X-ray

SNAP 25 P13795-2 77 neuronal fusion complex X-ray

SV 40 virus coat P03087 66 assembled coat X-ray

TAFII230 P51123 67 TBP NMR

TBS virus coat P11795 34 assembled coat X-ray

Tcf3 CAA67686* 41 -catenin X-ray

Tcf4 Q9NQB0 24 -catenin X-ray

Troponin I P19429 17 Troponin C NMR

Vitamin D3R P11473 89 DNA X-ray

Q3 SOV0

20

40

60

80

%

PSE: predictability of secondary structure

IDP

Partner

Fuxreiter et al. (2004) JMB 338, 1015

MorE, LM, PSE: devices of effective recognition

MoRE

PSE

Lacy et al (2004) NSMB 11, 358

Sequential mechanism of p27 binding

45

3) Tertiary structure

Dedmon et al. (2005) JACS 127, 476

Structural ensemble of a-synuclein

(NMR paramagnetic relaxation enhancement)

SAXS distance-distribution SAXS distance-distribution function and function and

topology of cellulase Etopology of cellulase E

Von Ossowski et al. (2005) Biophys. J. 88, 2823

102

103

104

105

106

107

Number of residues

Hy

dro

dy

na

mic

vo

lum

e, Å

3

Native

MGPMG

U (RC)

IUPPM

G

IUPRC

Uversky (2002) Prot. Sci. 11, 739

Global (tertiary) structure of IUPs

A lesson from denatured states of globular proteins:

Gillespie et al (1997) JMB 268, 170

spatial topology in denatured state resembles native structure (David

Shortle)

p27

ModelModelss

Protein trinity

Protein quartet

ordered

molten globule

random coil MG RC

ordered PMG

(Dunker) (Uversky)

The evolution of protein disorder

Evolution

Generation

Disorder in complete genomes (PONDR)

Dunker et al. (2000) Genome Inf. 11, 161

Disorder in complete genomes (DISOPRED)

Ward et al. (2004) JMB 337, 635

IDPs: high frequency in proteomes

Tompa et al. (2006) J. Prot. Res 5, 1996

coli

yeast

Structural disorder: evolutionary success story

20

40

60

0

LD

R (

40<

) pr

otei

n,

%

Domain of life

B

A

E

Vucetic et al. (2002) Proteins 52, 573

The evolution of protein disorder

Evolution

Generation

de novo generation

gene duplicationlateral gene transfer, LGT

The evolution of protein disorder

Evolution

Generation

Mutations

Point mutation

de novo generation

gene duplicationlateral gene transfer, LGT

Brown et al. (2002) J. Mol. Evol. 55, 104

Rapid evolution by point mutations

0

5

10

15

20

smallersamelargernu

mbe

r of

fam

ilies

evolutionary variability IUP vs glob.

Non-synonymous vs. synonymous substitutions

Point mutations

Synonymous (Ks)

Non-synonymous (Ka)

Nonsense

Evolution (Ka/Ks):

0.1-0.2: „functional”

1.0: „neutral” 1.0: „adaptive”

Rapid Rapid evolution of SRY of SRY genegeneSRY: sex determining region on the Y

chromosome (testis determining factor)

The evolution of protein disorder

Evolution

Generation

Mutations

Point mutation

Repeat expansion

de novo generation

gene duplicationlateral gene transfer, LGT

RNA polymerase II

TFs

Initiation

Elongation

Termination

CTDK

RNAP II CTD: coordination of 5’ capping, splicing, 3’ polyadenylation of mRNA

IGTGAFDVMIDEESLVKYMPEQKITEIEDGQDGGV

TPYSNESGLVNADLDVKDELMFSPLVDSGSNDAMA

GGFTAYGGADYGEATSPFGAYGEAPTSPGFGVSSP

GFSPTSPTYSPTSPAYSPTSPSYSPTSPSYSPTSP

SYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSP

SYSPTSPSYSPTSPSYSPTSPSYSPTSPSYSPTSP

SYSPTSPSYSPTSPAYSPTSPSYSPTSPSYSPTSP

SYSPTSPSYSPTSPNYSPTSPSYSPTSPGYSPGSP

AYSPKQDEQKHNENENSR

Yeast RNAP II CTDYeast RNAP II CTD

RNAP II CTD evolution

-2.5 -2.0 -1.5 -1.0 -0.5 0.0

10

20

30

40

50

60

time (GYr)

rep

eat

nu

mb

er

-SPSYSPT-

Repeats in IUPs and other datasets

0

5

10

15

20

25

30

35

40

Swiss-Prot Yeast Human IUP

freq

uenc

y (%

)

protein dataset

proteins

residues

Tompa (2003) BioEssays 25, 847

Protein

(repeat region)Repeat sequence Repetition Function Type

Calreticulin E/D2-8K/R1-3 6weak, large-capacity calcium

bindingI

Cdk p57 AP 43 linker between domains I

RS protein SC-35 (K)RS 50 mRNA splicing I

mastermind G1-7V/A 7 linker/spacer I

TF GAL11 Q 23assembly of transcription

preinitiation complexI

CPEB Q1-13R/I/L/S 15 regulation of mRNA translation I

Sup35p Q2X2NN/Y 14 Nonsense mutation suppression I

Sry (QQK)0,1Q2-13FHDH1-5 1– 19transactivator domain of sex-

determining factorI

Functional microsatellites (short repeats) in IDPs

MPRVYIGRLSYNVREKDIQRFFSGYGRLLEVDLKN

GYGFVEFEDSRDADDAVYELNGKELCGERVIVEHA

RGPRRDRDGYSYGSRSGGGGYSSRRTSGRDKYGPP

VRTEYRLIVENLSSRCSWQDLKDFMRQAGEVTYAD

AHKERTNEGVIEFRSYSDMKRALDKLDGTEINGRN

IRLIEDKPRTSHRRSYSGSRSRSRSRRRSRSRSRR

SSRSRSRSISKSRSRSRSRSKGRSRSRSKGRKSRS

KSKSKPKSDRGSHSHSRSRSKDEYEKSRSRSRSRS

PKENGKGDIKSKSRSRSQSRSNSPLPVPPSKARSVSPPPKRATSRSRSRSRSKSRSRSRSSSRD

SFRS6_HUMAN Splicing SFRS6_HUMAN Splicing factorfactor

MEGHVKRPMNAFMVWSRGERHKLAQQNPSMQNTEISKQLGCRWKSLTEAEKRPFFQEAQRLKILHREKYPNYKYQPHRRAKVSQRSGILQPAVASTKLYNLLQWDRNPHAITYRQDWSRAAHLYSKNQQSFYWQPVDIPTGHLQQQQQQQQQQQFHNHHQQQQQFYDHHQQQQQQQQQQQQFHDHHQQKQQFHDHHQQQQQFHDHHHHHQEQQFHDHHQQQQQFHDHQQQQQQQQQQQFHDHHQQKQQFHDHHHHQQQQQFHDHQQQQQQFHDHQQQQHQFHDHPQQKQQFHDHPQQQQQFHDHHHQQQQKQQFHDHHQQKQQFHDHHQQKQQFHDHHQQQQQFHDHHQQQQQQQQQQQQQFHDQQLTYLLTADITGEHTYQEHLSTALWLAVS

Mouse SRYMouse SRY (testis determining (testis determining factor)factor)

Protein

(repeat region)Repeat sequence Repetition Function Type

fibronectin-binding protein A (Du-D4)

EDT/SX9,10GGX3,4I/VDF

2 – 5 fibronectin binding I

involucrin (Q-region) QEGQLK/EH/LL/PEQ 24 – 63transglutaminase cross-

linking to form keratinocyte envelope

I

neurofilament-H (KSP domain)

XKSPY1-3K 42 – 55entropic sidearm of

neurofilamentsI

prion protein (octarepeats)

PQ/HGGGWGQ 3 – 14 copper binding III

RNA polymerase II (CTD)

YSPTSPS 11 – 52coordination of

transcription and mRNA processing

II

salivary PRPsPPPGKPQGPPPQGG

NKPQGPP6 – 33

binding of polyphenolic plant compounds

(tannins)I

tau proteinVQ/K/TSKI/CGSL/T/

KD/E/GNI/LK/H/THV/KQPGGG

3 – 5microtubule-binding,

polymerizationI

titin (PEVK)

PEV/APKEVVPEKKA/VPVAPPKKPEV/

APPVKV

5 – 60providing entropic elasticity during

sarcomere stretchI

Functional minisatellites (long repeats) in IDPs

MSQQHTLPVTLSPALSQELLKTVPPPVNTHQEQMKQPTPLPPPCQKVPVELPVEVPSKQEEKHMTAVKGLPEQECEQQQKEPQEQELQQQHWEQHEEYQKAENPEQQLKQEKTQRDQQLNKQLEEEKKLLDQQLDQELVKRDEQLGMKKEQLLELPEQQEGHLKHLEQQEGQLKHPEQQEGQLELPEQQEGQLELPEQQEGQLELPEQQEGQLELPEQQEGQLELPQQQEGQLELSEQQEGQLELSEQQEGQLELSEQQEGQLKHLEHQEGQLEVPEEQMGQLKYLEQQEGQLKHLDQQEQEGQLEQLEEQEGQLKHLEQQEGQLEHLEHQEGQLGLPEQQVLQLKQLEKQQGQPKHLEEEEGQLKHLVQQEGQLKHLVQQEGQLEQQERQVEHLEQQVGQLKHLEEQEGQLKHLEQQQGQLEVPEQQVGQPKNLEQEEKQLELPEQQEGQVKHLEKQEAQLELPEQQVGQPKHLEQQEKHLEHPEQQDGQLKHLEQQEGQLKDLEQQKGQLEQPVFAPAPGQVQDIQPALPTKGEVLLPVEHQQQKQEVQWPPKHK

INVO_HUMAN InvolucrinINVO_HUMAN Involucrin

................SDLGLCKKRPKPGGWNTGG

SRYPGQGSPGGNRYPPQGGGGWGQPHGGGWGQPHG

GGWGQPHGGGWGQPHGGGWGQGGGTHSQWNKPSKP

KTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPIIH

FGSDYEDRYYRENMHRYPNQVYYRPMDEYSNQNNF

VHDCVNITIKQHTVTTTTKGENFTETDVKMMERVV

EQMCITQYERESQAYYQRGSSMVLFSSPPVILLIS

FLIFLIVG

PRIO_HUMAN major prion protein

IUPsIUPs often evolve by repeat often evolve by repeat expansionexpansion

Basic mechanisms of repeat Basic mechanisms of repeat expansionexpansion

Meiotic: replication slippage (micro)

Mitotic: unequal crossing over (mini)

Wells RD (2001) JBC 271, 2875)

Replication slippageReplication slippage

(Unequal) crossing (Unequal) crossing overover

Morgan 1916

Evolution of repetitive regions in IUPs

Tompa (2003) BioEssays 25, 847

Type I

Type II

Type III