ebi is an outstation of the european molecular biology laboratory. protein function prediction from...

EBI is an Outstation of the European Molecular Biology Laboratory.

Protein Function Prediction From StructureIn Structural Genomics

Its Contribution To The Study Of Health And Disease

•James Watson

Erice 40th School of Crystallography

Structure To Function In Structural Genomics:Contribution To The Study Of Health And Disease

07/06/082

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07/06/083

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07/06/084

From Genome To Proteome?

• Determine every structure?

• Homology Modelling?

- Too expensive and time consuming

- Ultimately might be technically impossible?

- Find closest sequence match in PDB and use as start point to simulate structure

- Dependant on widespread fold coverage

Estimate of 20,000 to 25,000 genes in the human genome

Structural Genomics


07/06/085

Structural Genomics Aims

?

Pathogens and disease

Human proteins

Coverage of fold space

Automation / high throughput


07/06/086

Structural Genomics Collaborators

MCSG – Mid-west Centre for Structural Genomics

SPINE – Structural Proteomics in Europe

SGC – Structural Genomics Consortium


07/06/087

MCSG pipeline

Structural andStructural andFunctional Analysis

Functional Analysis

Automated C

rysta

l

Automated C

rysta

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Mountin

g and

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Structu

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eth

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Web Site

Web Site

Workshops

Workshops

Publications

Publications

Cleavage On Column

Cleavage On Column

New Tags andNew Tags andExpression SystemsExpression Systems

Targe

t Ref

inem

ent

Targe

t Ref

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Dom

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FUNCTIONALSTUDIES

GENOMICSEQUENCES


07/06/088

MCSG Collaboration


07/06/089

How Do We Define Function?

Question: What is the function of a cooker?

Burn Natural Gas?Boil water?

Grill fish?

Kitchen appliance?

Bake pie?

Central Heating?Gene OntologyEC nomenclature


07/06/0812

Function Prediction

Also Known As: Guess Who?

• Ask the right questions

• Ask enough questions


07/06/0813

A Friendly Warning!

when computers are applied to biology, it is vital to understand the difference between mathematical & biological significancecomputers don’t do biology

In short, you need to determine whether the information is reliable or not

they're often misleading & sometimes wrong!Don't always believe what databases tell you

they're sometimes misleading & occasionally wrong!Don't always believe what lecturers tell you

they're often misleading & sometimes wrong!

Don't always believe what programs tell you


07/06/0814

Methods

Evolutionary relationships

Metabolome

Genome organisation Biological

multimeric state

Electrostatics

OHH

NO

R R'

R''NH

O O R''

R R'

+

Catalytic clusters, mechanisms & motifs Ligands

Clefts and surfaces

MACiE


07/06/0815

Residueconservation

Sequence motifs(PROSITE, BLOCKS,SMART, Pfam, etc)

Sequence scans

Sequence search vs Uniprot and PDB

Gene neighbours

Superfamily HMMlibrary

Fold search (SSM and DALI)

Surface clefts

Nest analysis

Reverse templates

Structure scans

Templates

Enzyme active sites

Ligand binding sites

DNA binding sites

Laskowski RA, Watson JD & Thornton JM (2005).ProFunc: a server for predicting protein function from 3D structure.

Nucleic Acids Res., 33, W89-W93.


07/06/0816

Templates

GARTfaseCholesterol oxidaseIIAglc histidine kinase

Carbamoylsarcosineamidohhydrase

Dihydrofolate reductase Ser-His-Aspcatalytic triad


07/06/0817

Reverse Templates3-residue templates

2

4 5

87

3

6

9

…

1


07/06/0818

Template Matching – False Positives

Cambridge Erice Barcelona

Car Park

ChurchLibrary

High False Positive

Marsala

No False Positives


07/06/0819

Comparison Of Template Environments

Template structure – 1mbb

Arg

Glu

Ser

Match to template:

Query structure – 1hsk


07/06/0820


Arg

Glu

Ser

Match to template:




07/06/0821


Identical residues in neighbourhood:




07/06/0822


Arg

Glu

Ser

Similar residues in neighbourhood:




07/06/0823

Tempura Templates – User Refined Approach

X

X

X

Arg 295Asp296Gly 297Ala 298Gly 299His 300Tyr 301Gly 302

…

…

List of Structures

Single Structure

Templates

www.ebi.ac.uk/thornton-srv/databases/tempura/


07/06/0824

It can be hard to judge whether something “makes sense”.

The lack of labeling on many web pages makes it hard to know the source.

Calculations based on databases are even harder to deal with

Functional Annotation Transfer

???

Logical deductions may be worse.

“tacR gene regulates the human nervous system”

“tacQ gene is similar to tacR but is found in E. coli”

“so tacQ gene regulates the E. coli nervous system”


07/06/0825

It’s not all depressing though…..

“Sigh!”


07/06/0826

MCSG as a test dataset (1)Reference: Watson et al (2007), J.Mol.Biol. 367,1511-1522

282 non-redundant

UnknownFunction

PutativeFunction

KnownFunction

30% Seq ID

33% (93)

47%

20%

319 MCSGStructures

(Sep 2005)


07/06/0827

MCSG as a test dataset (1)

KnownFunction Results

BackdatingTop Hit

Manual Checking


07/06/0828

Manual Assessment of Methods

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

SIT

DNA

LIG

ENZ

SSM SSM protein fold match

Enzyme active site templates

Ligand-binding templates

DNA-binding templates

Reverse templates


07/06/0829

PDB entry: 1m33

• BioH protein from Escherichia coli • Contains Pfam domain

• PF00561: alpha/beta hydrolase fold common to a number of hydrolytic enzymes

Example 1: Predicted function confirmed

R.Sanishvili et al. (2003). Integrating Structure, Bioinformatics, and Enzymology to Discover Function. J. Biol. Chem. 278(28): 26039-26045.

• Search against the enzyme templates database provides a significant hit to Ser-His-Asp catalytic triad (rmsd = 0.28 Å)


07/06/0830

Example 1: Predicted function confirmed

Catalytic triad = lipase, protease, or esterase activity?

Serine nucleophile (Ser82) is located within one of the two Gly-Xaa-Ser-Xaa-Gly motifs present

= acyltransferase or thioesterase activity ?

Experimentally demonstrated carboxylesterase activity (EC 3.1.1.1).

A novel carboxylesterase with broad substrate specificity and a preference for short chain substrates.


07/06/0831

PDB entry: 2plm

• Tm0936 from Thermotoga maritima • Contains Pfam domain

• PF01979: Amidohydrolase family which contains a number of deaminases and is part of a wider Amidohydrolase superfamily clan.

Example 2: Identifying previously published function

J.C.Hermann et al. (2007). Structure-based activity prediction for an enzyme of unknown function. Nature, 448, 775-779.

• Publication suggests function: adenosine deaminase (E.C.3.5.4.4)

• Performed targeted docking of high-energy metabolic intermediates• Results dominated by adenine analogues undergoing C6-deamination• Structure determined with S -adenosylhomocysteine (SAH)• Clone provided by the JCSG

Method:


07/06/0832

2plm

1a4l

2plmTemplate Searches

1a4l

Sequence Identity = 17.5%, Local sequence identity = 27.7%. Structural Similarity = 95%

• Strong Enzyme template match (e-value = 2.45 E-04) • Structure: Adenosine deaminase (E.C.3.5.4.4)

Identifying previously published function


07/06/0833

Example 3: Putative Function

Function prediction: Metallo-beta-lactamase/nuclease

or phosphodiesterase

APC29563: Crystal structure of a hypothetical protein from Enterococcus faecalis V583.

Evidence:Sequence: Superfamily hit to metallo-hydrolase/oxidoreductaseBLAST hits to putative metallo-beta-lactamases

Fold, Ligand Templates (Zn) and Reverse Templates:Hits to metallo-beta-lactamase proteins and RNA degradation enzymes.

Some metallo--lactamases have been shown to have phosphodiesterase activity


07/06/0834

First pass screening

Phosphatase

Phosphodiesterase

Dehydrogenase

NADPH Oxidase

Oxidase

Protease Lipase

Thioesterase

Amino Acids AcidsSugarAlcoholAldehyde


07/06/0835

Phosphodiesterase Assay

0

0.5

1

1.5

2

2.5

3

3.5

4

2’3’ Cyclic mononucleotides are preferred substrates


07/06/0836

Detailed assays

1. Preferred metal • Cobalt gives strongest

activity

0

0.5

1

1.5

2

2.5

none Mg Mn Ni Co Zn Cu Ca

1.

2. Saturation curve • 2’3’ cAMP saturation curve calculated• Suggests kinetics:

i. Km near 1.2 mM

ii. Vmax near 2.9 mmol min-1 mg-1

0

0.5

1

1.5

2

2.5

3

0 1 2 3 4 5 6 71.

2. 3. No preference for 2’ or 3’ position

2’cAMP

3’cAMP

Adenosine

2’3’cAMP

Samples


07/06/0837

Putative Function

1. Shown phosphodiesterase activity against 2’3’ cyclic mononucleotides

2. Interestingly, is structurally similar to PDB entry 2dkf • Identified by fold and reverse template matches • Published as an RNA degradation protein of the metallo-beta-

lactamase superfamily

3. Possible RNA degradation protein?

More to be done……


07/06/0838

Problem Structures

Hypothetical protein from Bacillus subtilis (PDB entry 1Q8B)

1. Sequence Methods

2. Fold Comparison

3. Templates

4. Reverse templates

NAMSANDKLTILW

No Motifs

Hypothetical ProteinsHypothetical Proteins

Lots of Hits Hypothetical Proteins

Plant “Stable” Proteins

No Hits


07/06/0839

Medically relevant structuresStudying the molecular basis for ligand selectivity in a family of transcriptional regulators from Pseudomonas aeruginosa

Dimeric transcription factors which respond to small phenolic molecules and are responsible for antibiotic resistance.

13 PA sequences 5 structures solved, 1 with ligand bound

Implications to Cystic Fibrosis patients


07/06/0840

The Future Of Structural Genomics?

NIAID Category A, B, and C Priority Pathogens

Viral hemorrhagic fevers Toxoplasma Rabies


07/06/0841

Future Methods

Using binding site 3D atomic similarities to predict ligand binding &

Protein Function

CleftXplorer IsoCleft Finder

Abdullah Kahraman Rafael Najmanovich


07/06/0842

CleftXplorer - Algorithm for shape comparison

Kahraman, A., Morris, R. J., Laskowski, R. A. & Thornton, J. M. (2007). Shape variation in protein binding pockets and their ligands.

J Mol Biol 368, [email protected]


07/06/0843

2pd0: Cryptosporidium parvum protein, unknown function

•No sequence- or structure-based hints from Profunc•Bound MES (blue) used to define binding site

•Top 2 hits (red and green respectively) are analogs of the product and substrate of the same reaction in Humans and E.coli.

+ +Purine nucleoside phosphorylase:

Putative function:

[email protected]

IsoCleft Finder – query a database of binding sites


07/06/0844

Conclusions

- Need to use as many techniques as possible

- Successes but still need new methods and assays

- In ProFunc the fold and reverse templates most successful

HTP ligand binding assays, HTP enzyme assays, IsoCleft, CleftXplorer, etc.

EBI is an Outstation of the European Molecular Biology Laboratory.

EBI Resources And Services

Outreach and training

•James Watson


07/06/0846

Interactive training for all levels of experience1.Hands-on training in our purpose-built IT training

suite at EMBL-EBI, Hinxton, Cambridge2.EBI Roadshows bring expert trainers in our resources

to your site with a variety of modules on offer

3.New e-learning platform currently in development 4. Full programme at www.ebi.ac.uk/training/

Wellcome Images


07/06/0847

Coming up in our Hands-on Training2008

Patterns, similarities and differences in biological data

9–11 June

28–31 July Programmatic access of Proteomics ResourcesInteractions and Pathways26–27 August

1–3 September8–11 September

ENFIN Advanced Course on Protein Function PredictionProgrammatic access in Perl: webservices and workflowsA two-day dip into the EBI’s resources6–8 October

24–27 November

Programmatic access in Java: webservices and workflows

Transcriptomics resources and data analysis

19–22 January23–26 February16–18 March

27–29 April

Bioinformatics resources for protein structureSequence to genes: genome informatics

A walk through EBI Bioinformatics Resources

Programmatic access to biological databases

11–15 May

2009


07/06/0848

The Bioinformatics Roadshow 06/07

Stanford and UCSD, Jun 06

(all core

services)

Leuven, Oct 06

(MSD) Cambridge, Nov

06 (MSD)

Portsmouth,

Nov 06 (MSD)

Melbourne, Jan

07 (proteomics)

Harvard & MIT Mar 07 (all core

services)

Trieste (ICGEB, Jun 07 (all core

services)

Subscribe to the EBI-FELICS Roadshows calendar at http://www.google.com/calendar/

Liverpool, Apr 07 (modules

tbc)

Valencia, Apr 07 (modules tbc;

BioSapiens)

Oxford, Dec 06 (all core

services)

Exeter, Feb 07

(MSD)

Basel, Sep 07 (modules tbc;

BioSapiens)


07/06/0849

Roadshow modulesGenomes

Ensembl, EMBL-Bank, Integr8

GenomesEnsembl, EMBL-Bank, Integr8

TranscriptomesArrayExpress,

Expression Profiler,

R/Bioconductor

TranscriptomesArrayExpress,

Expression Profiler,

R/Bioconductor

ProteomesUniProt, InterPro,

IntAct, PRIDE, OLS

ProteomesUniProt, InterPro,

IntAct, PRIDE, OLS

StructuresMSD, PDBSum,

ProFunc

StructuresMSD, PDBSum,

ProFunc

PathwaysReactome, BioModels, BRENDA

PathwaysReactome, BioModels, BRENDA

Mini modulesWeb services; BioMart;

SRS; ChemistryGO/GOA; Alignments;

Literature

Mini modulesWeb services; BioMart;

SRS; ChemistryGO/GOA; Alignments;

Literature


07/06/0850

eLearning pilot project

Sequence searching1. Introduction2. BLAST for beginners3. Intermediate BLAST4. Patterns, profiles & HMMs5. Other tools: SSAHA,

FASTA, MPSrch

Sequence searching1. Introduction2. BLAST for beginners3. Intermediate BLAST4. Patterns, profiles & HMMs5. Other tools: SSAHA,

FASTA, MPSrch

For each module…1. Video tutorial2. Print tutorial3. Key concepts quiz4. Reflective tasks

For each module…1. Video tutorial2. Print tutorial3. Key concepts quiz4. Reflective tasks

More to come …1. Basic and advanced courses on

core data resources2. Web services3. Structural Biology Resources

More to come …1. Basic and advanced courses on

core data resources2. Web services3. Structural Biology Resources

Looking for beta-testers!


07/06/0851

Acknowledgements

• Funding: MCSG, NIH/NIGMS PSI, BioSapiens

• Structures: MCSG + many others

• Enzyme Assays:

Alexei Savchenko, Alexander Yakunin, Mike Proudfoot.

• Thornton Group

• EBI Outreach and Training Team

• Organisers and of course, you!

ebi is an outstation of the european molecular biology laboratory. protein function prediction from...

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