predicting the function of a protein form either a sequence or a structure (is not trivial) adam...

Predicting the function of a protein form either a sequence or

a structure(is not trivial)

Adam Godzik

The Sanford-Burnham Medical Research Institute

Summary - overview

Homology based methods Analogy based methods Physics based methods Why function prediction?

What we mean by function

Multilevel definition Phenotype Cellular function Molecular function

(activity) Substrates Inhibitors cofactors

Several attempts to develop a unified function classification EC classification for

enzymes 4.2.31.101

Merops (proteases), CAZY (hydrolases)

Gene ontology

Two, complementary views of the evolution and diversity of life

Organisms (species) Genes (proteins)

Both are amazingly large and diverse

Organisms (species)

About 1.5M known today, 10-100 million species estimated to exists, depending on the definition of species and other assumptions

Their relations can be described in a tree of life, at least for eukaryotes.

Bacterial and archeal tree of

life is much more controversial, some even dispute the concepts of species for bacteria

Proteins

With 20 amino acid alphabet, the number of possible protein sequences is very large (20100 i.e. 1.2*10130 short proteins(!))

Total number: >10billions? 10-100M species, with ~4K genes

in a bacterial and ~10K in an eukaryotic genome

Over 25 million known today, i.e. ~0.2%

Representative sample?

From the 25 million proteins known today

Direct experimental data is available for few thousand proteins

Indirect experimental data are available for perhaps few hundred thousand

Structures of ~60 thousands have been solved

protein universe seems to be very large. But is it random?

Many proteins (like species) are close relatives

Histone H1 (human) - histone H1 (chicken)

SRRSASHPTYSEMIAAAIRAEKSRGGSSRQSIQKYIKSHYKVGHNADLQIKLSIRRLLAA | | || || || ||| ||| | |||||||||||||||||| ||| |||||| || SKKSTDHPKYSDMIVAAIQAEKNRAGSSRQSIQKYIKSHYKVGENADSQIKLSIKRLVTT

similarity: 77% id, BLAST e.value 0.0 function: two H1 histones from

different species (orthologs) Their functions and structures are

obviously very similar

We can organize the protein universe into neighborhoods

(families)?

Number of protein clusters (modeling families) grows linearly in number of protein sequences (and exponentially in time) – cumulative total

Rate of discovery

0

50

100

150

200

250

0 1 2 3 4 5 6 7

Number of sequences (millions)

Nu

mb

er

of

clu

ste

rs (

tho

us

an

ds

)

size >=3

size >=5

size >=10

size >=20

From Yooseph et al, PloS Biology, (2007) 5:e16

How many protein families are still out there?

How far can we go?

Histone H5 - histone H1

TYSEMIAAAIRAEKSRGGSSRQSIQKYIKSHYKVGHNADLQIKLSIRRLLAAGVLKQTKGVGASGSFRLA | | | | | | | | | ||| | | | |||| |||||||| SVTELITKAVSASKERKGLSLAALKKALAAGGYDVEKNNSRIKLGLKSLVSKGTLVQTKGTGASGSFRLS

similarity: 40% seq id, BLAST e.value 10-15

function: two histones (paralogs) Structures still very similar,

functions somewhat different, but obviously similar

This is surely too far?

Histone H5 - TRANSCRIPTION FACTOR E2F-4

PTYSEMIAAAIRAEKSRGGSSRQSIQKYIKSHYKVGHNADLQIKLSIRRLLAAGVLKQTKGVGASGSFRL | | | | |

GLLTTKFVSLLQEAKD-GVLDLKLAADTLA------VRQKRRIYDITNVLEGIGLIEKKS----KNSIQW

similarity :7% seq id, BLAST e.value 1

Is it?

Structure – obviously similar (2.4 Å RMSD over 80 aa)

function – clearly related (both bind DNA)

More subtle similarity can be detected with more sophisticated methods

We can keep adding more layers

most “function assignments” are provided by predicted

homology

Unknown protein GLLTTKFVSLLQEAKDGVLD

LKLAADTLAVRQKRRIYDITNVLEGIGLIEKKSKNSIQW

Well studied protein SRRSASHPTYSEMIAAAIRAE

KSRGGSSRQSIQKYIKSHYKVGHNADLQIKLSIRRLLAASimilarity

->homology

prediction?

similarity

Similarity -> homology based annotations

Recognition of close and/or distant homologs based on similarity Sequence Sequence/profile,

profile/profile Structure

Problems How to predict

differences? Even homologous proteins evolve and change!

Prediction by homology

Recognition

Are there any well characterizedproteins similar to my protein?

Can we assume they are homologous?

Structure of my protein is similar to the other one

Modeling

Alignment What is the position-by-positiontarget/template equivalence

Function prediction

Function of my protein is similar to the other one

We could predict

activityRole in the

whole organism

3D structureStructure of a complex

Important distinction

Similarity Two proteins have similar

sequences/structures/functions if by some metric the s/s/f of one protein is more similar to the s/s/f of another than to a randomly chosen protein

Homology Two proteins are

homologous if they have evolved from a common ancestor

Common error Two proteins are 65% homologous

What we really meant The sequences of two proteins are 65% similar,

therefore we can safely assume they are homologous, why else they would be so similar?

If life would be easy, this is how it would look like

similarhomologous

not similarunrelated

Not (obviously) similar, but (probably) homologous

Histon H5 and transcription factor E2F4, identity 7%, similar fold, similar function (DNA binding)

PTYSEMIAAAIRAEKSRGGSSRQSIQKYIKSHYKVGHNADLQIKLSIRRLLAAGVLKQTKGVGASGSFRL | | | | |

GLLTTKFVSLLQEAKD-GVLDLKLAADTLA------VRQKRRIYDITNVLEGIGLIEKKS----KNSIQW

Similar, but not homologous

phosphoribosyltransferase and viral coat protein, identity: 42%, different folds, different functions

. . . . . 99 IRLKSYCNDQSTGDIKVIGGDDLSTLTGKNVLIVEDIIDTGKTMQTLLSLVRQY.NPKMVKVASLLVKRTPRSVGY 173 : ||. ||| || |. || | : | | | | || | || |:| | ||.| |214 VPLKTDANDQ.IGDSLY....SAMTVDDFGVLAVRVVNDHNPTKVT..SKVRIYMKPKHVRV...WCPRPPRAVPY 279

Similarity vs. homology

similarhomologous

not similarunrelated

not similarhomologous

similarnot homologous

Can we return to this simple picture by redefining

similarity?

similarhomologous

not similarunrelated

Are these two protein families related?

New protein (target)

KAAELEMEKEQILRSLGEISVHNCMFKLEECDREEIEAITDRLTKRTKTVQVVVETPRNEEQKKALEDATLMIDEVGEMMHSNIEKAKLCLQ

Known protein (template)

VKKDALENLRVYLCEKIIAERHFDHLRAKKILSREDTEEISCRTSSRKRAGKLLDYLQENPKGLDTLVESIRREKTQNF

How to compare two families?

alignment Fam Fam jmijli BMMA1 2Score = ?

Compare asvectors in 21dimensionalspace (FFAS)

Profile-profile similarity

How to validate a protocol1. Recognition

Folding benchmarks from structural clustering of PDB (several

sets, 700 pairs used here)compared to sequence based clustering of the same group of proteins

correct predictions vs. wrong predictions CASP meetings, CAFASP, LiveBench published and/or publicly available

predictions, fold prediction servers, available prediction programs

Summary - overview

Homology based methods Analogy based methods Physics based methods Why function prediction?

Similarity -> analogy based annotations

Recognition of potential analogs based on similarity in Genome organization

(non homologous replacements)

Genomic fingerprints Expression patterns Specific features

Charge distribution Presence of specific

patterns

Problems Is this similarity

related to function?

TM0449 (thy1) – from prediction to proof

TM0449 Hypothetical, uncharacterized

protein Multiple homologs in

pathogenic and thermophilic bacteria

Novel fold evidence

Phylogenetic profile complementing thymidylate synthase

A homolog complements TS in Dictyostelium

Confirmed experimentally

3D motif search finds an identical arrangement binding phosphate in a

different protein

Summary - overview

Homology based methods Analogy based methods Physics based methods Why function prediction?

“Ab initio function prediction” – substrate docking

We know the structure of one protein in the family and functions of

some others – is the function conserved?

Newly solvedtarget

Gallery of models

We can analyze conservation of surface features by mapping them

on the sphere

And then compare maps between homologs

And come up with new (predicted) functions

Phospholipid vs. retinol vs. short peptide binding

Summary - overview

Homology based methods Analogy based methods Physics based methods Why function prediction?

Why my interest in function prediction?

Structural genomics: the structure is often the easiest experimental information to obtain (after sequence)

Function vs function

We witnessed dramatic technological advances in sequencing and now structure determination, function analysis remain a painstaking, manual effort.

We used to know a lot about function even before we started working on a protein. Well, not anymore

1990 2005 2010 ?

1 y

ear

Structure determination

1970

Function discovery

Sequencing

purificationexpressioncloning

struc. refinementstruc. validationannotationpublication

phasingdata collectionxtal screening tracingbl xtal mounting

crystallizationimagingharvesting

targetselection

3 X 2 X 5 X

1 X1 X 1 X

2 X

1 X 1 X 2 X 2 X 1 X

7 X1 X

1 X

1 X

PDB

1 X

Structure determination is now done on an assembly

line

purificationexpressioncloning

struc. refinementstruc. validationannotationpublication

phasingdata collectionxtal screening tracingbl xtal mounting

crystallizationimagingharvesting

targetselection

3 X 2 X 5 X

1 X1 X 1 X

2 X

1 X 1 X 2 X 2 X 1 X

7 X1 X

1 X

1 X

PDB

1 X

Even few years ago functional annotation

seemed trivial

purificationexpressioncloning

struc. refinementstruc. validationannotationpublication

phasingdata collectionxtal screening tracingbl xtal mounting

crystallizationimagingharvesting

targetselection

1 X

2 X 2 X 1 X

7 X1 X

1 X

1 X

PDB

1 X

After few years, the reality seems to be very different

“reverse order” of function and structure determination and it’s challenges

The classical way 1. A function is discovered

and studied 2. The gene responsible in

this function is identified 3. Function is confirmed 4. Product of this gene is

isolated, crystallized solved.

5. we have a whole story!

Structure “rationalizes” function and provides molecular details

Post-genomic 1. a new, uncharacterized gene

is found in a genome 2. predictions or high-

throughput methods prioritize this gene for further studies

3. the protein is studied in detail

Structure is solved in a high throughput center

Structure is the first experimental information about the “hypothetical” protein

We now have hundreds of structures of proteins with

unknown functions

Summary

For some, function prediction is a practical, day to day problem

Analogy based approaches dominate the field Homology seen from sequence similarity structural similarities Potential active sites, clefts, surface features

Many useful tools exists, but they are very scattered and not very user-friendly

Summary (2)

Avoid overconfidence - “easy” predictions contain many surprises

Only synergy of several independent lines of reasoning can give a correct answer

Elimination of “easy”, but inconsistent predictions is critical

So far, AFP doesn’t even come close to expert analysis