Protein structure in molecular systems biology

McKim Conference on Predictive McKim Conference on Predictive Toxicology, Duluth MN, Sept. 25-Toxicology, Duluth MN, Sept. 25-27th 200727th 2007

Protein structure in Protein structure in molecular systems biologymolecular systems biology

Dr DAVID SHEEHAN, Dr DAVID SHEEHAN,

PROTEOMICS RESEARCH GROUP,PROTEOMICS RESEARCH GROUP,

DEPT. BIOCHEMISTRY, DEPT. BIOCHEMISTRY,

UNIVERSITY COLLEGE CORK,UNIVERSITY COLLEGE CORK,

IRELANDIRELAND

http://www.ucc.ie/


THE ECOTOXICOLOGY THE ECOTOXICOLOGY TRIANGLETRIANGLE

BIOTA ENVIRONMENT

POLLUTANTS

SEASONALITYCLIMATE

SPECIATIONDISTRIBUTION

TOXICOLOGYBIOTRANSFORMATIONBIOCONCENTRATION


SENTINEL SPECIES IN SENTINEL SPECIES IN ECOTOXICOLOGYECOTOXICOLOGY

• CHOSEN AS REPORTERS OF CHOSEN AS REPORTERS OF STATUSSTATUS

• ROBUSTROBUST

• EASILY-RECOGNISEDEASILY-RECOGNISED

• ABUNDANTABUNDANT

• SESSILE RATHER THAN MOBILESESSILE RATHER THAN MOBILE

• WIDE GEOGRAPHICAL WIDE GEOGRAPHICAL DISTRIBUTIONDISTRIBUTION


SENTINELS - SENTINELS - ADVANTAGESADVANTAGES• HISTORY OF USEHISTORY OF USE

• MULTI-ORGAN MODEL SYSTEMSMULTI-ORGAN MODEL SYSTEMS

• PREDICT FOR TOXICITY IN HUMANSPREDICT FOR TOXICITY IN HUMANS

• OFTEN BIOCONCENTRATE OFTEN BIOCONCENTRATE POLLUTANTSPOLLUTANTS


SENTINELS - SENTINELS - DISADVANTAGESDISADVANTAGES

• MAY HAVE UNIQUE UPTAKE MAY HAVE UNIQUE UPTAKE CHARACTERISTICSCHARACTERISTICS

• MUCH SIMPLER SYSTEMS THAN MUCH SIMPLER SYSTEMS THAN MAMMALSMAMMALS

• POORLY-REPRESENTED IN POORLY-REPRESENTED IN SEQUENCE D-BASESSEQUENCE D-BASES


MUSSELS – GENUS MUSSELS – GENUS MYTILUSMYTILUS• FILTER FEEDERSFILTER FEEDERS

• SESSILESESSILE

• ABUNDANTABUNDANT

• WIDELY-DISTRIBUTEDWIDELY-DISTRIBUTED

• RESILIENT TO POLLUTIONRESILIENT TO POLLUTION

• HISTORY OF USEHISTORY OF USE

• ““MUSSELWATCH” PROGRAMMESMUSSELWATCH” PROGRAMMES


SYSTEMS BIOLOGYSYSTEMS BIOLOGY

DNA mRNA PROTEIN METABOLITES

PROTEIN-PROTEININTERACTIONS

MODIFIEDPROTEINS

PHOSPHOPROTEOMEGENOME

TRANSCRIPTOME GLYCOPROTEOME

REDOX PROTEOME

PROTEOME

METABOLOME


MYTILUS IS POORLY-REPRESENTED MYTILUS IS POORLY-REPRESENTED IN D-BASESIN D-BASES

NUMBER OFNUMBER OF

ENTRIES (NCBI)ENTRIES (NCBI)MYTILUSMYTILUS MOUSEMOUSE

GENESGENES ~ 30,000~ 30,000 33,00033,000

ESTEST 29,10229,102 926,165926,165

PROTEINPROTEIN

SEQ.SSEQ.S1,7681,768 139,018139,018

STRUCTURESTRUCTURE

(PDB)(PDB)99 1,2621,262


Beta-1,4-D-Endoglucanase Cel45a From Blue Mussel Mytilus Edulis

X-Ray Structure Of Beta-Mannanase From Blue Mussel Mytilus Edulis

Crystal Structure Of Phosphoenolpyruvate Mutase – 6 str.s

Mediterranean Mussel Defensin Mgd-1

http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi/mmdb.cn3/?cmdMmdb=View3D&Complexity=3&dopt=j&save=Viewer&uid=38921





SOME IMPLICATIONS OF POOR D-SOME IMPLICATIONS OF POOR D-BASE REPRESENTIONBASE REPRESENTION

• SPOT-MATCHING FOR MS IS SPOT-MATCHING FOR MS IS DIFFICULTDIFFICULT

• LACK OF STRUCTURAL KNOWLEDGE LACK OF STRUCTURAL KNOWLEDGE THROUGH PROTEIN HIERARCHYTHROUGH PROTEIN HIERARCHY

• DIFFICULT TO LOCATE PROTEIN DIFFICULT TO LOCATE PROTEIN MODIFICATIONS (e.g. OXIDATION)MODIFICATIONS (e.g. OXIDATION)

• PROTEIN-PROTEIN INTERACTION?PROTEIN-PROTEIN INTERACTION?


OVERVIEW OF THIS OVERVIEW OF THIS PRESENTATIONPRESENTATION

• SEQUENCE-INDEPENDENT PROBES SEQUENCE-INDEPENDENT PROBES FOR REDOX LESIONS IN PROTEINSFOR REDOX LESIONS IN PROTEINS

• SEQUENCE SIMILARITY IN SPOT SEQUENCE SIMILARITY IN SPOT MATCHINGMATCHING

• USE OF ON-LINE HOMOLOGY USE OF ON-LINE HOMOLOGY MODELSMODELS


SEQUENCE-SEQUENCE-INDEPENDENT PROBES INDEPENDENT PROBES FOR REDOX LESIONS IN FOR REDOX LESIONS IN

PROTEINSPROTEINS


Oxidative stressOxidative stress

ANTIOXIDANTANTIOXIDANT

DEFENCEDEFENCEROSROS

H202

.OOR .

OH

OH-.O2

-

O2+

CAT GPXSOD

GSH


PROTEIN EXPRESSION SIGNATURE APPROACHPROTEIN EXPRESSION SIGNATURE APPROACH

pH

70 kDa

10

Mr

17 kDa

3

UP-REGULATED

DOWN-REGULATED

NO CHANGE

IMAGE ANALYSIS

SOFTWARE


PROTEIN OXIDATION PROTEIN OXIDATION COMPLICATES THE PROTEOMECOMPLICATES THE PROTEOME

• PROTEINS ABSORB PROTEINS ABSORB ~ ~ 70% OF ROS70% OF ROS

• CARBONYLATION (R CARBONYLATION (R → → ALDEHYDE/KETONE)ALDEHYDE/KETONE)

• UBIQUITINATIONUBIQUITINATION

• NITROSYLATIONNITROSYLATION

• CYS OXIDATION ….CYS OXIDATION ….

Dowling & Sheehan D. (2006) Proteomics 6, 5597-5604.Sheehan D. (2006) Biochemical and Biophysical Research Communications 349, 455-462.


PROTEIN PROTEIN CARBONYLATION:ANIMALS CARBONYLATION:ANIMALS EXPOSED TO PRO-OXIDANTSEXPOSED TO PRO-OXIDANTS

Blot Name Blot Name (Anti-DNP)(Anti-DNP)

SpotSpotss

MatMatchedched

MatcMatch h RateRate

*Menadione *Menadione GillGill

3333 3333 100%100%

Control GillControl Gill 1616 1313 81%81%

HH22OO22 Gill Gill 6464 2828 40%40%

CdCCdCl2l2 Gill Gill 3737 2525 67%67%

1 2

3 4

4 7pI4 7pI

66

45

31

21.5

66

45

31

21.5

Mr

(x10

-3)

MYTILUS EDULIS 2-D CARBONYLS__________________________

McDonagh & Sheehan, 2006

Aquat. Toxicol. 79, 325-333

C = O HYDRAZINE - DNP+ BLOT

ANTI-DNPHYDRAZONE - DNP


REDOX LESIONS: REDOX LESIONS: UBIQUITINYLATIOUBIQUITINYLATIONN

__________________________

McDonagh & Sheehan, 2006

Aquat. Toxicol. 79, 325-333

Ub

Ub

ANTI-UbUb

Ub

Ub

UbBLOT


REDOX LESIONS: REDOX LESIONS: NITROSOTHIOLS NITROSOTHIOLS AND THE AND THE BIOTIN SWITCH ASSAYBIOTIN SWITCH ASSAY• NITROSOTHIOLS ARE REDUCED BY ASCORBIC ACID NITROSOTHIOLS ARE REDUCED BY ASCORBIC ACID

BUT DISULPHIDES ARE NOT – SPONTANEOUSLY BUT DISULPHIDES ARE NOT – SPONTANEOUSLY HYPERTENSIVE HYPERTENSIVE RAT KIDNEY MEDULLA RAT KIDNEY MEDULLA

SNO SHSS SSASCORBIC ACID

NEM - BIOTIN

S-NEM-BIOTINSSSTREPTAVIDIN


REDOX LESIONS: SULPHENIC ACID REDOX LESIONS: SULPHENIC ACID (SOH) PROTEINS(SOH) PROTEINS

SH-S – S-

SOH

S-NEM -S – S-

SOHNEM

S-NEM -S – S-

SHARSENITE

DTT

S-NEM -SH

SOH

SH-

PROTEINEXTRACT

ATS

S-NEM

-SH

SOH

SH-

AFFINITY SELECTION

FLOW-THROUGH

2DE

. ...

.

IMAGE ANALYSIS

2D LC-TANDEM MS

IDENTIFICATION

NEM - BIOTIN


TOXICOLOGY OF NANOPARTICLES TOXICOLOGY OF NANOPARTICLES IN IN E. COLIE. COLI – – GOLD AS ANTIOXIDANTGOLD AS ANTIOXIDANT

PROTEIN GOLD

-SH- IODOACETAMIDEFLUORESCEIN

MENADIONE CONTROL


REDOX LESIONS: REDOX LESIONS: NITROSOTYROSINESNITROSOTYROSINES

YNOSSANTI -3NITROTYROSINE

YNOSS

LC-TANDEM MS

IDENTIFIED 22 PROTEINSTyther et al., 2007 Proteomics –

Clinical Applications. IN PRESS


SEQUENCE SIMILARITY IN SEQUENCE SIMILARITY IN SPOT MATCHINGSPOT MATCHING


Cork harbour

7 days

24 hr+/- 1 mM

menadione

DISSECT GILL

PROTEINS

O

O

CH3

http://images.google.it/imgres?imgurl=http://www.iol.ie/~discover/orksee_files/cross1.jpg&imgrefurl=http://www.iol.ie/~discover/orksee.htm&h=321&w=452&sz=24&hl=it&start=13&um=1&tbnid=tXdlRsNobXB9KM:&tbnh=90&tbnw=127&prev=/images%3Fq%3Dcork%2Bharbour%2Bireland%2Bmap%26svnum%3D10%26um%3D1%26hl%3Dit%26sa%3DN

http://images.google.it/imgres?imgurl=http://geology.com/world/satellite-image-of-ireland.jpg&imgrefurl=http://geology.com/world/ireland-satellite-image.shtml&h=930&w=759&sz=232&hl=it&start=66&um=1&tbnid=JVagvfFLlYriKM:&tbnh=147&tbnw=120&prev=/images%3Fq%3Dcork%2Bharbour%2Bireland%2Bmap%26start%3D63%26ndsp%3D21%26svnum%3D10%26um%3D1%26hl%3Dit%26sa%3DN


Control Menadione

A

B

hspgp96

PDI

calreticulinhmb

ProteaseSerine 1

45 kDa

PDI

betatubulin

alpha 2tubulin

gelsolinenolase

GDP diss. inhibitor

GST Pi

RNA binding protein

transferrn

45 kDa

-SH “DISAPPEAR”

-S-S “APPEAR”

ON OXIDATION….-SH + IAF -S-IAF


SPOT IDENTIFICATIONSPOT IDENTIFICATION

TRYPTICDIGEST

HPLC

MS 1 MS 2

TANDEM MS (i.e. MS/MS)

PROTEIN PEPTIDES PEPTIDE 1 m/z FRAGMENT n m/z

AMINO ACIDSEQUENCE

SCREEN SEQD-BASESSPOT ID.


31Cell controlMus musculus (Mouse)Protease serine 19*

No identification8

34Tetraodon nigroviridis (pufferfish)Unamed7*

319Heavy metal bindingM. edulisHeavy metal binding protein

6


5

227Heavy metal bindingM. edulis (Blue mussel)Heavy metal binding protein

4

69Binds misfolded proteinsMeloidogyne incognita (cotton root-knot nematode)

Calreticulin3

149Protein foldingConus marmoreus(Cone Snail)PDI2

260ChaperoneStrongylocentrotus purpuratus (Sea Squirt)

Heat shock protein gp961

MOWSEFunctionSpeciesProtein NameSpot no.

31Cell controlMus musculus (Mouse)Protease serine 19*

No identification8

34Tetraodon nigroviridis (pufferfish)Unamed7*


6


5

227Heavy metal bindingM. edulis (Blue mussel)Heavy metal binding protein

4

69Binds misfolded proteinsMeloidogyne incognita (cotton root-knot nematode)

Calreticulin3

149Protein foldingConus marmoreus(Cone Snail)PDI2

260ChaperoneStrongylocentrotus purpuratus (Sea Squirt)

Heat shock protein gp961

MOWSEFunctionSpeciesProtein NameSpot no.

McDonagh & Sheehan, 2007,

Proteomics, 7, 3395-3403


USE OF ON-LINE USE OF ON-LINE HOMOLOGY MODELSHOMOLOGY MODELS


PROCEDURE FOR PROCEDURE FOR HOMOLOGY MODELLINGHOMOLOGY MODELLING

TEMPLATE

STRUCTURE

TARGET

SEQUENCE

IDENTIFY STRUCTURALLY

CONSERVED REGIONS

CREATE DATABASE OF LOOPS

FROM ELSEWHERE IN PDB

SCRs

LOOPS

ENERGY MINIMISE

HOMOLOGY MODEL


COMBET et al., “Geno3D an automated protein modelling web server, BIOINFORMATICS, 2002, 18, 213-214 http://pbil.ibcp.fr/htm/

ON-LINE HOMOLOGY MODELLING WITH ON-LINE HOMOLOGY MODELLING WITH Geno3DGeno3D


ON-LINE HOMOLOGY ON-LINE HOMOLOGY MODELLING WITH Geno3DMODELLING WITH Geno3D

• START AT PROTEIN SEQUENCE DATABASE START AT PROTEIN SEQUENCE DATABASE

• AMINO ACID SEQUENCE IS INPUT FOR ON-AMINO ACID SEQUENCE IS INPUT FOR ON-LINE HOMOLOGY MODELING PROGRAM e.g. LINE HOMOLOGY MODELING PROGRAM e.g. GENO3D.GENO3D.

• SELECT TEMPLATESELECT TEMPLATE(PSI-BLAST)(PSI-BLAST)

• RESULTS SENT RESULTS SENT VIA e-MAIL.VIA e-MAIL.


CATALASE FROM CATALASE FROM M. CalifornianusM. Californianus

TEMPLATE: HUMAN ERYTHROCYTE CATALASE PDB 1F4J


CATALASE FROM CATALASE FROM M. CalifornianusM. Californianus


MODEL MODEL QUALITYQUALITY


STRUCTURALSTRUCTURALANALYSISANALYSIS

MAIN CHAIN PARAMETERS ARECOMPARED TO A STRUCTURE

OF 2Å RESOLUTION


ROOT MEAN SQUARE DEVIATIONROOT MEAN SQUARE DEVIATION

-Cs

TEMPLATE

MODEL

x1x2

x3

x4

x5

X6 .. RMSD = x2

n


PROTEINPROTEIN

(% Id)(% Id)

TARGETTARGET

(ACCESSION )(ACCESSION )

TEMPLATETEMPLATE

(PDB ID)(PDB ID)

RMSDRMSD

((ÅÅ) (% Id)) (% Id)

RAMACHANDRANRAMACHANDRAN

(% DISALLOWED)(% DISALLOWED)

MODELMODEL

HISTONE H3HISTONE H3 AAP94664AAP94664 1KX51KX5 6.86 (98.5)6.86 (98.5) 4.24.2

CYT. C CYT. C OXIDASEOXIDASE

AAT98405AAT98405 1V54 11.01 (58.4)11.01 (58.4) 1.11.1

TROPO-TROPO-MYOSINMYOSIN

BAA19209BAA19209 2TMA2TMA 23.94 (50.9)23.94 (50.9) 6.16.1

00.10.20.30.40.50.60.70.80.9

11.11.21.31.41.5

0 20 40 60 80 100

% sequence identiy betweenthe target and the template

RMSD IS ONLY LOOSELY RELATED TO % SEQ. ID.


RAMACHANDRAN RAMACHANDRAN ANALYSISANALYSIS

CN

AMIDE PLANE

SER ARG

PSIPHI

D:\McKim Presn\spdbv\spdbv\spdbv.exe


LACTOPEROXIDASE: OVERLAY OF LACTOPEROXIDASE: OVERLAY OF MODEL ON TEMPLATE (PyMOL)MODEL ON TEMPLATE (PyMOL)

MOVIE!!

MODEL TEMPLATE


OTHER MODELSOTHER MODELS

METHIONINE ADENOSYLMETHYL TRANSFERASE

CYTOCHROME COXIDASE

HEAVYMETAL B.P.

LYSOZYME MDRASSOC.DPROTEIN

P-450 P-450 CYP4Y1CYP4Y1

PHOSPHOFRUCTOKINASE

P-GLYCOPROTEIN

SUPEROXIDEDISMUTASE

TWITCHIN


RELEVANCE OF HOMOLOGY RELEVANCE OF HOMOLOGY MODELSMODELS

• SIGNAL TRANSDUCTION -> SIGNAL TRANSDUCTION -> “NETWORKS OF PROTEINS”“NETWORKS OF PROTEINS”

• GUIDE FOR STRUCTURE-FUNCTIONGUIDE FOR STRUCTURE-FUNCTION• MODEL SOLVENT ACCESSIBILITYMODEL SOLVENT ACCESSIBILITY• PROTEIN-PROTEIN INTERACTIONSPROTEIN-PROTEIN INTERACTIONS• CAVEATS!!CAVEATS!!

– MODELS ARE NOT STRUCTURESMODELS ARE NOT STRUCTURES– SIDE-CHAINSSIDE-CHAINS


CONCLUSIONSCONCLUSIONS

• PROTEIN MODIFICATIONS NEED PROTEIN MODIFICATIONS NEED TO BE MODELLEDTO BE MODELLED

• MORE SEQUENCE DATA ARE MORE SEQUENCE DATA ARE REQUIRED FOR NON-STANDARD REQUIRED FOR NON-STANDARD SPECIESSPECIES

• THREE-DIMENSIONAL MODELS THREE-DIMENSIONAL MODELS ARE POSSIBLE WHERE 3D ARE POSSIBLE WHERE 3D STRUCTURE ARE LACKING STRUCTURE ARE LACKING


FUNDING/ACKNOWLEDGEMENTSFUNDING/ACKNOWLEDGEMENTS

• Prog. Res. Third Level Institutions; Prog. Res. Third Level Institutions; IRCSET; EPA; Health Research BoardIRCSET; EPA; Health Research Board

• Collaborators: Dr Tim Veenstra (NCI, Collaborators: Dr Tim Veenstra (NCI, NIH); Drs Ian Davidson, Phil Cash NIH); Drs Ian Davidson, Phil Cash (Aberdeen); Prof Maria Bebiano (Aberdeen); Prof Maria Bebiano (Faro)(Faro)

• Drs. Vera Dowling, Brian McDonagh, Ray TytherDrs. Vera Dowling, Brian McDonagh, Ray Tyther

• Sara Tedesco, Ebenezer Kanagaraj, Hu WentaoSara Tedesco, Ebenezer Kanagaraj, Hu Wentao

Protein structure in molecular systems biology

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