Multiple alignment of protein structures based on ligand or - - PowerPoint PPT Presentation

Multiple alignment of protein structures based

• n ligand or prosthetic group position

Jean-Christophe Nebel

Faculty of Computing, Information Systems & Mathematics Kingston University, London

j.nebel@kingston.ac.uk

http://www.kingston.ac.uk/~ku33185/Nestor3D.html

Some challenges

• Protein 3D structure prediction

(John Moult, organiser of CASP 6, December 2004)

Proteins with homologues: good models, but fine grain (all atoms) models are still

needed Twilight zone: approximate (and useful) models; further improvements will require full atom description and refinements

• Protein function annotation from 3D structure

Structural genomics projects: high-throughput delivery of protein structures regardless of the state of their functional annotation

• Drug design

High resolution models of active sites are required

Principles

• Active sites are key to understanding of protein functions
• Most conserved regions of homologue proteins are linked to

active sites (e.g. PROSITE patterns)

• Multiple alignment improves pattern recognition

(e.g. ClustalW)

Multiple alignment of protein 3D structures based on active site position at atomic level generation of 3D motifs

3D motif generation

• Rigid alignment of protein 3D structures based on local

features linked to active sites prosthetic groups

ligands PROSITE patterns (under development)…

• Generate consensus pattern based on threshold

atom positions (1-1.4 Å)

chemical group positions, e.g. carboxyl, (1.5-3.8 Å) cavity position, if relevant, (1-1.4 Å)

Validation: Proteins w ith porphyrin rings

• The PDB holds 1551 proteins containing these groups

(i.e. 5.3% of PDB entries, 01/02/05) globines peroxidases cytochromes P450s chlorophyll proteins…

• All atoms of the porphyrin ring used to align sets of

homologue proteins

Validation: Methodology

• Representatives of proteins containing porphyrin rings

PDB50% Identical chains and chains that are not involved with a prosthetic group were removed. Structures of 237 chains

• For each chain

Generation of a set of homologues (within our set) using FASTA Generation of a 3D template based on homologues only Comparison between template and PDB structure

Number of true and false positives Distribution of true positives

66 patterns w ere produced

(at least 3 homologues were required, E value of 10e-6, atom distance of 1.25 Å)

Validation: Results

Similar results with other parameters, groups and cavity Detection of abnormal structures: 1U5U & 1S05

1S05: structural model validated using a restricted set of NMR experiments 1U5U: protein fragment

True positives False positives

Application: Modelling of active site of CYP17

• P450 protein involved in biosynthesis of sex hormones
• Enzyme associated to some forms of cancer (breast & prostate)
• 3D structure is unknow n
• P450 active site:

Haem group linked to protein by a cysteine Ligand (i.e. drug) on the other side of the haem group

Homologues of p450 human CYP17

P450s: 3500+ sequences (50+ human genes) 125 structures ( 5 humans)

18 29.5% 417 1AKD 35 23.9% 417 1IZO 61 26.4% 389 1N97 63 24.0% 455 1H5Z 79 29.3% 455 1BU7 135 28.2% 485 1W0G 136 28.6% 476 1PQ2 Hits Identity% Length Protein

No single good candidate! But 5 proteins are close…

Consensus structure of CYP17

Haem group Cavity area Consensus atoms

Is it biologically meaningful?

Consensus groups

P450 know n patterns based on sequences

4 clusters based on structure the 4 most common patterns! e.g. blue box, P450 signature

[FW]-[SGNH]-x-[GD]-x-[RKHPT]-x-C-[LIVMFAP]-[GAP]

Modelling of P450 active site based on consensus 3D structures, J.-C. Nebel, International Conference on Biomedical Engineering, BioMed 2005, 16-18 February 2005, Innsbruck, Austria

Comparison w ith CYP17 active site models

Generated independently by Dr S. Ahmed*

(Kingston University - School of Chemical & Pharmaceutical Sciences)

*Ahmed S: The use of the novel substrate-heme complex approach in the derivation of a representation of the active site of the enzyme complex 17alpha-hydroxylase and 17,20-lyase. Biochem Biophys Res Commun 2004, 316(3):595-598.

Putative H-bond

Clustering according to active site similarity

(Kulczynski’s metric & Neighbour Joining) NESTOR3D ClustalW

Tow ards function prediction

Protein families: P450s Globines Flavocytochromes Peroxidases Catalases, Cytochromes B, C, C2, C3 & C’

Nestor3D: free softw are (Java)

• Functionality

Generation of consensus structures Generation of cavity descriptions Generation of similarity matrices

• Applications

Better understanding of active sites (drug design…) Function prediction: detection of putative active sites from a protein 3D structure generation of phylogenetic tree from similarity matrix Homology modelling generation of a structure template (constraints…) validation of predicted protein structure http://www.kingston.ac.uk/~ku33185/Nestor3D.html

• New method to produce high resolution active site models
• Consensus structure elements biologically meaningful and

related to function

• Require proteins interacting w ith ligands or heterogeneous

groups: rigid molecules:

6% PDB entries semi rigid molecules: 20% PDB entries

Conclusion Future w ork

• Alignments based on PROSITE patterns
• Generation of a 3D motif database

5 kinases (ATP or ADP)