1. From Structure to Function
Janet Thornton
European Bioinformatics Institute

2. From Structure to Functional Annotation

3. Mid-West Center for Structural Genomics (MCSG)
University of Toronto
Aled Edwards
Argonne National Laboratory
Andrzej Joachimiak
Northwestern University
Wayne Anderson
EBI / University College London
Janet Thornton, Christine Orengo
University of Washington at St Louis
Daved Fremont
University of Virginia
Wladek Minor
UT Southwestern Medical Center
Zbyszek Otwinowski

5. 60 structures solved to date
~30% are ‘hypothetical proteins’
Some examples …
ylxR hypothetical cytosolic protein
Hypothetical protein (EC4030_F)
Hypothetical protein (MTH1)
ygbM hypothetical protein (EC1530)
Conserved hypothetical protein (MT777)
cutA protein implicated in Cu homeostasis (TM1056)

6. TIM barrel enzymes – 18 different homologous families >60 different E.C. numbers
Structure of TIM barrel:
Triose phosphate isomerase
EC Wheel of TIM barrels

7. Pairwise sequence identity and conservation of enzyme function (Todd et al 2001)
Single-domain proteins: >81,000 homologous enzyme / enzyme and enzyme / non-enzyme pairs
Fractional
percentage

8. From Structure To Biochemical Function
Gene  Protein  3D Structure  Function
Given a protein structure:
Where is the functional site?
What is the multimeric state of the protein?
PQS – Hannes Ponstingl (this morning)
Which ligands bind to the protein?
What is biochemical function?

9. Automated Structure Comparison
The most powerful method for assigning function from structure is global or partial 3D structure comparison (e.g. Dali, SSAP; SSM)
Hidden Markov Models derived from structural domains can often recognise distant relatives from sequence
Christine Orengo (tomorrow)

10. Aspartate Amino Transferase Superfamily
Tyrosine Phenolyase
2,2-Dialkylglycine Decarboxylase
Ornithine Decarboxylase

11. Aspartate Amino Transferase Superfamily77 11 73 6 76 10 7 76
Tyrosine Phenolyase
Aspartate Aminotransferase 9 79 7 77
2,2-Dialkylglycine Decarboxylase
Ornithine Decarboxylase

12. Aspartate Amino Transferase Family
2,2-Dialkylglycine Decarboxylase
all bind Pyridoxal 5’ Phosphate (PLP) co-factor
Ornithine Decarboxylase
Tyrosine Phenolyase
Aspartate Aminotransferase

13. Number of enzyme functions
TIM barrel glycosyl
hydrolases
/ hydrolases
type I PLP-dependent enzymes

14. Convergent and Divergent Evolution
Unrelated proteins can perform the same function (convergent evolution), sometimes using the same mechanism – sometimes using different mechanisms
Related proteins can perform different functions – divergent evolution

15. Active site convergence
Trypsin
Subtilisin

16. Trypsin
Subtilisin
Alpha/beta hydrolase
CheB methylesterase
Clp protease
Brain platelet activating factor acetylhydrolase

17. Predicting Binding Site
Binding-site analysis: cutA
Surface clefts
Residue conservation
Conserved surface patches
Most likely binding site

18. Identifying Binding Site Function Using Motifs
- 3D enzyme active site structural motifs (Craig Porter)
- Catalytic Site Atlas - Identification of catalytic residues (Gail Bartlett, Alex Gutteridge)
- Metal binding sites (Malcolm MacArthur)
- Binding site features (Gareth Stockwell)
- Automatically generated templates of ligand-binding and
- DNA binding motifs (Sue Jones, Hugh Shanahan)
- “Reverse” templates (Roman Laskowski)
JESS – fast template search algorithm (Jonathan Barker)
PINTS - Searches for similar clusters (Aloy, Russell … – EMBL Heidelberg))

19. Catalytic Site Atlas
Enzyme reports from primary literature information
-lactamase Class A
EC:
PDB: 1btl
Reaction: -lactam + H2O  -amino acid
Active site residues: S70, K73, S130, E166
Plausible mechanism:

20. 3-D templates
Use 3D templates to describe the active site of the enzyme
analogous to 1-D sequence motifs such as PROSITE, but in 3-D
Sequence position independent
Captures essence of functional site in protein

21. TEmplate Search and Superposition TESS
Wallace et al., 1997
defines a functional site as a sequence-independent set of atoms in 3-D space
search a new structure for a functional site
search a database of structures for similar clusters
e.g. serine proteinase,
catalytic triad

22. Pepsin

23. Aspartic Proteinase - Active Site residues - [DTG]x2
Eukaryotic & Fungal Aspartic Proteinases:
all-atom DTG-DTG Template

24. Aspartic Proteases: Active Site Template
Asp CO2
Gly C
A template of 8 atoms
is sufficient to identify
all Aspartic Proteinases
Asp O
Gly C
Thr/Ser O
Thr O

25. Aspartic Protease Template Search against all PDB
green= true
red=false

26. 3D Templates to Characterise Functional Sites
Template searches
(189 enzyme active site templates)
(~600 Metal binding site templates)

27. Database of enzyme active site templates
Cholesterol oxidase
GARTfase
IIAglc histidine kinase …
Carbamoylsarcosine
amidohhydrase
Ser-His-Asp
catalytic triad
Dihydrofolate reductase

28. An example MCSG structure BioH – unknown function
involved in biotin synthesis
in E.coli
Structure: Rossmann fold, hence many
structural homologues
Expected to be an enzyme
Sequence contains two Gly-X-Ser-X-Gly motifs typical of
acyltransferases and thioesterases

29. CSA template search One very strong hit
Ser-His-Asp catalytic triad of the lipases with rmsd=0.28Å
(template cut-off is 1.2Å)
One very strong hit
Experimentally confirmed by hydrolase assays
Novel carboxylesterase acting on short acyl chain substrates

30. Templates of Active Sites
Catalytic cluster conserved – Simple template
e.g. Aspartic Proteinase (DTG)x2
Order and geometry of catalytic residues varies
Multiple templates e.g. Polymerases
Same catalytic cluster used in many different enzyme functions – one template identifies multiple active sites in unrelated structures
eg Asp/His/Ser catalytic triad is well conserved in structure

31. Instances of convergence
Ser-His-Asp triads
Cys-His-Asp triads
Ribonuclease T1s
Malic enzyme and isocitrate dehydrogenase
Haloperoxidases
Creatinase and carboxypeptidase G2
Glycosidases
Class II extradiol-type dioxygenase and class III extradiol-type dioxygenase
Receptor tyrosine phosphatase and low-molecular weight tyrosine phosphatase
Pyridoxal 5' phosphate enzymes
James Torrance

32. Template databases HAND CURATED
Enzyme active sites (PROCAT) – 189 templates
Currently being extended
Metal-binding sites – 600 templates
AUTOMATED
Ligand-binding sites – 10,000 templates
DNA-binding sites – 800 templates

33. Another example of convergent evolution: The DNA HTH Binding Motif
1b9m
1eto
1hcr
1ais
Sue Jones
1jhg
1lmb
1orc

34. ProFunc – function from 3D structure
Homologous sequences of known function
Functional sequence motifs
Q-x(3)-[GE]-x-C-[YW]-x(2)-[STAGC]
Homologous structures of known function
DNA-, ligand- binding and “reverse” templates
Enzyme active site 3D-templates
Binding site identification and analysis
Residue conservation analysis
HTH-motifs Electrostatics Surface comparison
… etc

35. Three MCSG Examples (James Watson)
Three examples show the varying levels of information that can be retrieved from structures:
1. Almost full functional information. GOOD
APC 1040
2. General information. NOT SO GOOD
APC 012
3. Little or no information obtained. UGLY
APC 078

36. Acknowledgements
Roman Laskowski, James Watson, Richard Morris, Rafael Najmanovich, Fabian Glaser - EBI
Christine Orengo, Annabel Todd, James Bray, Russell Marsden – University College, London
MCSG members – Andzrej Jaochimiak, Al Edwards etc
Funding: NIH - PSI; EU - SPINE; DoE – DNA Motifs; UK BBSRC LINK