A new approach towards deciphering the protein code: The protein assembly model

Claire Lesieurlesieur@lapp.in2p3.fr

NucleusNucleus ProteinProtein

LipidsLipids

ChromosomeChromosome

DNADNA

NucleusNucleus(chromosome)(chromosome)

Membrane (Lipids)Membrane (Lipids)

ProteinsProteins

CHONCHON

- CuttingCutting- RecognitionRecognition- EnzymeEnzyme- SignalingSignaling- CarrierCarrier- Shape generatorShape generator- Road networksRoad networks

Function Shape How the shape provides a particular

function How the shape is acquired

GKKHDGATTYQWGKKHDGATTYQW

????

How it folds: Mechanisms of protein folding

How the information is encrypted in the sequences: CODING problem

ADRTGGILLKMHGGARECVVP

All the information necessary for the protein folding is All the information necessary for the protein folding is within the protein primary sequencewithin the protein primary sequence

C.B. Anfinsen, Haber, E., Sela, M. & White, F. H. , Proc. C.B. Anfinsen, Haber, E., Sela, M. & White, F. H. , Proc. Nati. Acad. Sci. USA 47 (1961) 1309-1314.Nati. Acad. Sci. USA 47 (1961) 1309-1314.

Levinthal’s paradox(1968): not random search but directedLevinthal’s paradox(1968): not random search but directed

Levinthal, C. (1968) J. Chim. Phys. 65, 44-45.Levinthal, C. (1968) J. Chim. Phys. 65, 44-45.

Short range interactionStructure TertiaireStructure Tertiaire

COOHCOOH

HH22NNmsms s-hourss-hours

Structure primaireStructure primaire Structure SecondaireStructure Secondairelong-rangelong-rangeinteractionsinteractions

short-rangeshort-rangeinteractionsinteractions

X-ray crystallography + NMR: PDBX-ray crystallography + NMR: PDB

3D modeling: PDB3D modeling: PDB

~ 70 % Sequence similarity: 3D modeling~ 70 % Sequence similarity: 3D modeling

70 % similarity: different shape70 % similarity: different shape

Low sequence similarity: similar shapeLow sequence similarity: similar shape

Amino acids on the surface of proteins: Amino acids on the surface of proteins: changeablechangeable

-strands transmembrane domain: 1010101-strands transmembrane domain: 1010101-helice transmembrane domain: 11111111111111111-helice transmembrane domain: 11111111111111111

Geometrical constrain

Chemical constrain

Domains Shape and role

??Sequence Pattern

Sequence PatternSequence Pattern

Trends in Microbiology (2000). Vol 8 (4):169-172Trends in Microbiology (2000). Vol 8 (4):169-172

Cholera toxinCholera toxin

• ABAB5 5 toxintoxin– A catalytic subunitA catalytic subunit– B receptor binding subunitB receptor binding subunit

• GMGM11: cell receptor: cell receptor• Endocytosed and traffic to the Endocytosed and traffic to the

ERER• ADP ribosylation of GADP ribosylation of G subunit subunit• Increase of cAMP leading to Increase of cAMP leading to

water losswater loss

CtxBCtxB55

CtxACtxA

ERER

Assembly in vitroAssembly in vitro

PentamerePentamere

MonomereMonomere

pH 1pH 1

15 min15 min

pH 7pH 7

-1,2 10-1,2 1066-1 10-1 10

66-8 10-8 10

55-6 10-6 10

55-4 10-4 10

55-2 10-2 10

5500

2 102 1055

200200 210210 220220 230230 240240 250250

pH 1pH 1

pH 7pH 7

NativeNative

Wavelength (nm)Wavelength (nm)

Mea

n r

esid

ue

Mo

lecu

lar

Ell

ipti

city

Mea

n r

esid

ue

Mo

lecu

lar

Ell

ipti

city

Trp-fluorescence

Flu

ore

scen

ce I

nte

nsi

tyF

luo

resc

ence

In

ten

sity

320 340 360 3800

100

200

300

unfoldedunfolded

Wavelength (nm)

0

50

100

150

200

250

300

350

0 20 40 60

Time (min)Time (min)

exex= 295 nm= 295 nm

emem=352 nm=352 nm

Flu

ore

scen

ce I

nte

nsi

ty (

a.u

.)F

luo

resc

ence

In

ten

sity

(a.

u.)

His

0

20

40

60

80

100

4,5 5 5,5 6 6,5 7 7,5 8

pHpH

CtxBCtxBF

un

ctio

nF

un

ctio

n

HISTIDINEHISTIDINE

…

…CtxB5CtxB5

Heat labile enterotoxin BHeat labile enterotoxin B Cholera toxin BCholera toxin B

LTBLTB CtxBCtxB

N-terminal

0

20

40

60

80

100

4,5 5 5,5 6 6,5 7 7,5 8

pHpH

LTBLTBCtxBCtxB

Fu

nct

ion

Fu

nct

ion

N-terminalN-terminal

LTB5LTB5

Kinetics differences

On pathway intermediates differences

It is particular amino acids that are responsible for each individual step of assembly and folding

Alzheimer, Parkinson, Prion diseases

Protein X: FOLD state: healthyProtein X: FOLD state: healthy

(Protein X)n: Assembly state: Lethal(Protein X)n: Assembly state: Lethal

Information for interfacesInformation for interfaces

Protein Interface formation

Rules? Mechanism?

Preferential geometries related to preferential sequences of amino acids?

INTERFACES:Zone de contact entre monomeres voisins

Interface Trimer pentamer heptamer

Brin 1

Brin 2

0101 0101 Ch111Ch

n.a. Ch111Ch 1111/1

Fibritin like domain

Nombre de monomerNombre de monomer 2 3 4 5 6 7 8 9 10 11 12 2 3 4 5 6 7 8 9 10 11 12

Nombre de casNombre de cas 5722 1035 2340 168 721 46 512 45 87 8 2055722 1035 2340 168 721 46 512 45 87 8 205

513 -524 LMITTECMVTDL aaa-bbbbbbb-

35-49GRNVVLDKSFGAPTI--bbbb-------bb

Monomer M

Monomer M+ 1

Distances

5 97

1 92

2 525

177 367

1 194

4 233

96 336

2 526

6 523

64 549

3 97

1 94

344 471

8 80

5 111

10 88

1 302HY6 (30)

1N9R (68) 19 86

1WNR (94)

1JBM (78)

2F86 (129)

1G31 (107)

1LNX (74)

1Q57 (483)

2RAQ (94)

1GRL (518)

1IOK (524)

1PZN (240)

1J2P (229)

1Y7O(194)

2F6I (189)

1TG6 (193) 1 193

2CBY (179) 15 194

1OEL (525)

1LEP (92)

3BDU (51) 2 53

1HX5 (92)

betaalpharc

3BDU 20-29, 38-533BDU 20-29, 38-53

3BDU 1--111011-110110--103BDU 1--111011-110110--101G31 0--1-1001-100100--001G31 0--1-1001-100100--001JBM 110010001011001011011JBM 110010001011001011011LNX 1--0100010110000---11LNX 1--0100010110000---11N9R 0--0100011110010--111N9R 0--0100011110010--111J2P ----1000101100101--11J2P ----1000101100101--11HX5 ------0011110010--111HX5 ------0011110010--111LEP 0---10001000--00--111LEP 0---10001000--00--11Con2 ----1-001-1100-0-Con2 ----1-001-1100-0-

1LEP: 1-8, 88-94, 40-57

1WNR: 1-8, 88-94, 44-57, 62-77

1HX5: 5-11, 94-97, 51-62, 68-80, 27-30

1G31: 8-15, 104-111, 68-85

yeast

P. aerophilum: bacterium

Methanobacterium Thermautriophicum: extremophile

1N9R

1LNX

1JBM

1JBM: 12-18, 42-50, 64-83

1LNX: 10-15, 25-32, 40-48, 63-77

1N9R: 66-82yeast

P. AerophilumHyperthermophilic bacterium

Methanobacterium Thermautriophicum: extremophile

1

1 + 1

1 +1 +1

2CBY2CBY

Geometry and function related

Family of protein interfaces

Assembly keys

Classification of protein interfaces: Database

Systematic analysis of protein interfaces-subjective classification

Mathematical approach: Laurent Vuillon (LAMA)

Functional analysis of protein interfaces Protein Assembly mechanism from block:

Giovanni Feverati Stoechiometry/Symmetry: Paul Sorba Experimental tests: Claire Lesieur

Alicia Ng Ling

Mun Keat Chong Boon Leng Chua Danyang Kong

Giovanni Feverati Paul Sorba