Applications of Homology Applications of Homology ModelingModeling

Hanka Venselaar

This seminar….

Homology Modeling…• What?• Why?• When?• How?

• And a few real world examples….

MSQSTQTNEFLSPEVFQHIWDFLEQPICSVQPIDLNFVDEPSEDGATNKIEISMDCIRMQDSDLSDMWPQYTNLGLLNSMDQQIQNGSSSTSPYNTDHAQNSVTAPSPYAQPSSTFDALSPSPAIPSNTDYPGPHSFDVSFQQSSTAKSATWTYSTELKKLYCQIAKTCPIQIKVMTPPPQGAVIRAMPVYKKAEHVTEVVKRCPNHELSREFNEGQIAPPSHLIRVEGNSHAQYVEDPITGRQSVLVPYEPPQVGTEFTTVLYNFMCNSSCVGGMNRRPILIIVTLETRDGQVLGRRCFEARICACPGRDRKADEDSIRKQQVSDSTKNGDGTKRPFRQNTHGIQMTSIKKRRSPDDELLYLPVRGRETYEMLLKIKESLELMQYLPQHTIETYRQQQQQQHQHLLQKQTSIQSPSSYGNSSPPLNKMNSMNKLPSVSQLINPQQRNALTPTTIPDGMGANIPMMGTHMPMAGDMNGLSPTQALPPPLSMPSTSHCTPPPPYPTDCSIVSFLARLGCSSCLDYFTTQGLTTIYQIEHYSMDDLASLKIPEQFRHAIWKGILDHRQLHEFSSPSHLLRTPSSASTVSVGSSETRGERVIDAVRFTLRQTISFPPRDEWNDFNFDMDARRNKQQRIKEEGE

Sequence:

EEC syndrome

No structure:

EEC syndrome

Homology modeling in short…Prediction of structure based upon a highly similar structure

2 basic assumptions:

•Structure defines function

•During evolution structures are more conserved than sequence

Use one structure to predict another

Homology modeling

Example: by 80 residues 30% identity sufficient

# residues

% id

entit

y

*

* Actually, modelling is possible, but we cannot get an alignment…

O

Homology modeling in short…Prediction of structure based upon a highly similar structure

Add sidechains, Molecular Dynamics simulation on model

Unknown structure

NSDSECPLSHDG

NSDSECPLSHDG

|| || | ||

NSYPGCPSSYDG

Model sequence

Known structure

Known structure Back bone copiedCopy backbone and conserved residues

Model!

The 8 steps of Homology modeling

1: Template recognition and initial alignment

1: Template recognition and initial alignment

• BLAST your sequence against PDB

• Best hit normally template

• Initial alignment

NSDSECPLSHDGYCLHDGVC

|| || | ||||| |||

NSYPGCPSSYDGYCLNGGVC

1: Template recognition and initial alignment

2: Alignment correction

2: Alignment correction

• Functional residues conserved• Use multiple sequence alignments• Deletions shift gaps

CPISRTGASIFRCW CPISRTGASIFRCWCPISRTA---FRCW CPISRT---AFRCW

CPISRTAAS-FRCWCPISRTG-SMFRCWCPISRTA--TFRCWCPISRTAASHFRCWCPISRTGASIFRCW CPISRTA---FRCW

Both are possible

Multipe sequence alignment

Correct alignment

Sequence with known structure

Your sequence

2: Alignment correction

• Core residues conserved• Use multiple sequence alignments• Deletions in your sequence shift gaps

Known structure FDICRLPGSAEAV

Model FNVCRMP---EAI

Model FNVCR---MPEAI

S

G

P

L

A

E

R

C

I V

C

R

M

P

EV

C

R M

P

E

Correct alignment

F-D--A-V

1: Template recognition and initial alignment

2: Alignment correction

3: Backbone generation

3: Backbone generation

• Making the model….• Copy backbone of template to model• Make deletions as discussed• (Keep conserved residues)

1: Template recognition and initial alignment

2: Alignment correction

3: Backbone generation

4: Loop modeling

4: Loop modeling

Known structure GVCMYIEA---LDKYACNC

Your sequence GECFMVKDLSNPSRYLCKC

Loop library,

try different options

1: Template recognition and initial alignment

2: Alignment correction

3: Backbone generation

4: Loop modeling

5: Sidechain modeling

5: Side-chain modeling

• Several options• Libraries of preferred rotamers based

upon backbone conformation

1: Template recognition and initial alignment

2: Alignment correction

3: Backbone generation

4: Loop modeling

5: Sidechain modeling

6: Model optimization

6: Model optimization

• Molecular dynamics simulation• Remove big errors

• Structure moves to lowest energy conformation

1: Template recognition and initial alignment

2: Alignment correction

3: Backbone generation

4: Loop modeling

5: Sidechain modeling

6: Model optimization

7: Model validation

7: Model Validation

• Second opinion by PDBreport /WHATIF• Errors in active site? new alignment/

template

• No errors? Model!

1: Template recognition and initial alignment

2: Alignment correction

3: Backbone generation

4: Loop modeling

5: Sidechain modeling

6: Model optimization

7: Model validation

8: Iteration8: Iteration

8: Iteration

8: Iteration

Model!

1: Template recognition and initial alignment

2: Alignment correction

3: Backbone generation

4: Loop modeling

5: Sidechain modeling

6: Model optimization

7: Model validation

8: Iteration8: Iteration

8: Iteration

8: Iteration

8 steps of homology modeling1: Template recognition and initial alignment2: Alignment correction3: Backbone generation4: Loop modeling5: Side-chain modeling6: Model optimization7: Model validation8: Iteration

Alignment

Modeling

Correction

MSQSTQTNEFLSPEVFQHIWDFLEQPICSVQPIDLNFVDEPSEDGATNKIEISMDCIRMQDSDLSDMWPQYTNLGLLNSMDQQIQNGSSSTSPYNTDHAQNSVTAPSPYAQPSSTFDALSPSPAIPSNTDYPGPHSFDVSFQQSSTAKSATWTYSTELKKLYCQIAKTCPIQIKVMTPPPQGAVIRAMPVYKKAEHVTEVVKRCPNHELSREFNEGQIAPPSHLIRVEGNSHAQYVEDPITGRQSVLVPYEPPQVGTEFTTVLYNFMCNSSCVGGMNRRPILIIVTLETRDGQVLGRRCFEARICACPGRDRKADEDSIRKQQVSDSTKNGDGTKRPFRQNTHGIQMTSIKKRRSPDDELLYLPVRGRETYEMLLKIKESLELMQYLPQHTIETYRQQQQQQHQHLLQKQTSIQSPSSYGNSSPPLNKMNSMNKLPSVSQLINPQQRNALTPTTIPDGMGANIPMMGTHMPMAGDMNGLSPTQALPPPLSMPSTSHCTPPPPYPTDCSIVSFLARLGCSSCLDYFTTQGLTTIYQIEHYSMDDLASLKIPEQFRHAIWKGILDHRQLHEFSSPSHLLRTPSSASTVSVGSSETRGERVIDAVRFTLRQTISFPPRDEWNDFNFDMDARRNKQQRIKEEGE

P63 Structure!

EEC syndrome

EEC syndrome

Arginine

Serine

Mutation RS

•Loss of negative charge

•Loss of interaction with the DNA

Another real world example:Another real world example:

Mutation analysis HFEMutation analysis HFE

HFE – complex:

HFE

β2-microglobulin Facilitates trafficking of HFE to the cellmembrane

Transferrin receptor (dimer)binds iron/transferrin complex

-Signaling and regulation of iron in bloodstream.

-Expressed in liver and colon.

-Mutations cause iron deposition disease “Hereditary Hemachromatosis“

Hereditary Hemachromatosis3 occuring mutations

C280Y

D41HL161P

•C280Y

•D41H

•L161P

Mutation C260Y

•Loss of cystein bridge

•Disturbing of β2-microglobulin binding domain

•No trafficking to membrane

Mutation H41D

•Introduction of additional negative charge

•Disturbing of hydrogen bridges

•Loss of stability in this area

Mutation L161P

•Loss hydrophobic interactions

•Major disturbance of the helix

•Less interaction of the helix with the transferrin receptor

Seriousness of mutation

D41H

L161P

C260Y

Seriousness of the disease

D41H

L161P

C260Y

Conclusion: the seriousness of the mutation is related to the seriousness of the disease and can be explained by analyzing the mutations with the 3D structure.

Homology Modeling…• What? Prediction of an unknown structure based on an

homologous and known structure• Why? To answer biological and medical questions when the

“real” structure is unknown• When? A template with enough identity must be available• How? 8 Steps

• Real world examples: mutations in EEC syndrome and HFE can be explained

To conclude….