Comparing and Classifying Domain Structures Methods for comparing protein structures Protein structural classifications How do structures and functions diverge in protein superfamilies What proportion of genome sequences can be predicted to belong to superfamilies of known structure?

Protein Domain Family Classifications Known domain structures Alexey Murzin, LMB, Cambridge Predicted domain structures Julian Gough, Bristol University Known domain structures Predicted domain structures Christine Orengo, UCL Domain sequences Alex Bateman, Sanger

domains are important evolutionary units 60-80% of genes in genomes code for multidomain proteins

Evolution gives rise to families of proteins (homologues) Domain Superfamily human yeast human M. tuberculosis Th. thermophilus structure is more highly conserved than sequence during evolution At least 40-50% of the structure is conserved 4

Evolution gives rise to families of proteins (homologues) orthologues Domain Superfamily human yeast human M. tuberculosis Th. thermophilus structure is more highly conserved than sequence during evolution At least 40-50% of the structure is conserved 5

Evolution gives rise to families of proteins paralogues Domain Superfamily human yeast human M. tuberculosis Th. thermophilus structure is more highly conserved than sequence during evolution At least 40-50% of the structure is conserved 6

Structural diversity in the CATH Domain Family P-loop hydrolases Cocaine esterase Acetylcholinesterase Cutinase structure is more highly conserved than sequence during evolution At least 40-50% of the structure is conserved

Challenges in comparing protein structures residue substitutions due to single base mutations insertions or deletions (indels) of residues - usually not in the secondary structures but in the connecting loops Usually the structural cores are highly conserved Although structure is much more conserved than the sequence there can still be considerable structural differences between relatives outside the core

residue insertions usually occur in the loops connecting secondary residue insertions usually occur in the loops connecting secondary structures substitutions can cause shifts in the orientations of secondary structures

Superposition of OB fold Structures

Related structures RMSD usually < 3.5A

Coping with Insertions and Deletions ignore the variable loop regions and only compare the secondary structures use algorithms which can explicitly handle insertions/deletions e.g. dynamic programming, simulated annealing

Fast structure comparison by secondary structures

In this example the common graph contains 5 nodes. Graphs can be compared using the Bron Kerbosch algorithm to find the largest common graph In this example the common graph contains 5 nodes. E E E E E E H E H H H H H H H H H Generallly ~1000 times faster than residue based methods

STRUCTAL Score distances between superposed residues in path matrix Use dynamic programming to find best path Align sequences Superpose structures Use equivalences given by the best path to re-superpose the structures

Structure Comparison Algorithms Structure classification Secondary structure based: SSM Henrick PDB GRATH Harrison & Orengo CATH Residue based: SSAP Taylor and Orengo CATH DALI Holm and Sander SCOP Comparer Sali and Blundell HOMSTRAD FatCat Adam Godzik PDB Structal Levitt PDB Structural Bioinformatics, Ed: Phil Bourne, Wiley 2003 Bioinformatics: Genes, Proteins and Computers, Bios, 2003

Domain structure database lass Domain structure database A Orengo & Thornton 1993 rchitecture T opology or Fold Group H omologous Superfamily ~200,000 domains 2600 domain superfamilies

C A T H 3 ~40 ~1200 ~200,000 domains Class Architecture Topology or Fold 3 ~40 ~1200 domain database

CATH Architectures Orthogonal bundle Up-down bundle -horseshoe a-solenoid aa-barrel b-ribbon b-sheet b-roll b-barrel

CATH Architectures Clam 2-layer b-sandwich Trefoil Orthogonal b-prism Parallel b-prism 3-layer b-sandwich b-solenoid ab-roll b-propeller

CATH Architectures ab-barrel 2-layer (ab) sandwich 3-layer (aba) sandwich 3-layer (bba) sandwich 3-layer (bab) sandwich 4-layer (abba) sandwich ab-prism ab-box ab-horseshoe

C A T H ~200,000 domain entries 40,000 domain entries Topology or Fold Group ~1200 40,000 domain entries ~200,000 domain entries Homologous Superfamily ~2600 Sequence Family (30%)

Divergent Evolution Convergent Evolution Divergent Evolution ..VILST… ..KLST… ...SLTRF... ..VILST… ..KLST… ...SLTRF... Convergent Evolution Convergent Evolution

Homologous Structures cholera toxin pertussis toxin SSAP score 97 81 79% 12% Sequence identity Heat labile enterotoxin high structure similarity score, often < 4A may have detectable sequence similarity e.g. by HMMs related functions

Evolutionary Ancestry Uncertain structural similarity no sequence similarity no functional similarity Evolutionary Ancestry Uncertain

How do proteins evolve new functions?

Evolution of Protein Functions in Domain Superfamilies domain duplication residue mutations and domain structure embellishments domain fusion, change in domain partner oligomerisation

Mutation of Residues TIM barrel glycosyl hydrolases acid chitinase A Glu general acid narbonin Glu incorporated in a salt-bridge and this blocks substrate access

Changes in domain function in paralogous relatives EC code: 2.7.7.3 2.7.7.39 binding site binding site Pantetheine-phosphate adenyltransferase Glycerol-3-phosphate cytidylyl transferase changes in the domain structure can modify the binding site or domain surface

Pantetheine-phosphate adenyltransferase Arginyl-tRNA synthetase binding site Pantetheine-phosphate adenyltransferase Arginyl-tRNA synthetase 1od6A00 1f7uA01

Arginyl-tRNA synthetase

changes in the domain partnerships can modify the binding site Pantetheine-phosphate adenyltransferase Asparagine synthetase B

Change in Oligomerisation Thioredoxin superfamily peroxidase calsequestrin

The Mosaic Theory of Protein Evolution Teichmann et al 2001,2003 Gerstein et al. 2001 60-80% of proteins are multi-domain few thousand domain superfamilies (< 10,000 CATH, SCOP and Pfam) > Two million domain combinations (multi-domain architectures)

Similarity in Chemistry conserved I P 19% P semiconserved I 67% P P P poorly conserved I 7% P P I’ P’ 7% unconserved nearly 90% of families show full or partial conservation of functions

chemistry is conserved or semi-conserved across the family but the substrate can change cytochrome P450s FAD/NAD(P)(H)-dependent disulphide oxidoreductases hexapeptide repeat proteins

blade domain

fulcrum domain 41

handle domain 42

How representative are these structural superfamilies (ie in CATH, SCOP) of all proteins in nature?

:Domain structure predictions in genome sequences protein sequences from UniProt scan against library of sequence patterns (HMM models) for CATH ~ 26 million domain sequences assigned to CATH superfamilies ~6000 annotated genomes

10,340 curated families with annotation Pfam-A Pfam-B Other Pfam-A 10,340 curated families with annotation 47

CATH and Pfam coverage of genomes NewFam?

Protein Family Databases Each family is represented by a sequence profile or HMM