From Structure to Function

Janet Thornton European Bioinformatics Institute

From Structure to Functional Annotation

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 UT Southwestern Medical Center Zbyszek Otwinowski University of Virginia Wladek Minor

60 structures solved to date

~30% are ‘hypothetical proteins’ Some examples …

ylxR hypothetical cytosolic protein ygbM hypothetical protein (EC1530) Hypothetical protein (MTH1) Hypothetical protein (EC4030_F) Conserved hypothetical protein (MT777) cutA protein implicated in Cu homeostasis (TM1056)

TIM barrel enzymes – 18 different homologous families >60 different E.C. numbers

Structure of TIM barrel: Triose phosphate isomerase EC Wheel of TIM barrels

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 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 0 10 1 1 20 21 -3 0 31 -4 0 41 -5 0 51 -6 0 61 -7 0 71 -8 0 81 -9 0 91 -1 00 Sequence identity (%) Unconserved Conserved

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?

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)

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

Aspartate Amino Transferase Superfamily 77

2.6.1.1

Aspartate 76 Aminotransferase 10 6 73 11 9 79 7 77 7 76

4.1.99.2

Tyrosine Phenolyase

4.1.1.64

2,2-Dialkylglycine Decarboxylase

4.1.1.17

Ornithine Decarboxylase

Aspartate Amino Transferase Family

4.1.1.64

2,2-Dialkylglycine Decarboxylase

4.1.1.17

Ornithine Decarboxylase all bind Pyridoxal 5’ Phosphate (PLP) co-factor

4.1.99.2

Tyrosine Phenolyase

2.6.1.1

Aspartate Aminotransferase

20 10 0 60 50 40 30

Number of enzyme functions

TIM barrel glycosyl hydrolases  /  hydrolases type I PLP-dependent enzymes structural data structural and sequence data superfamilies

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

Active site convergence

Trypsin Subtilisin

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

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

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))

Catalytic Site Atlas

Enzyme reports from primary literature information –  -lactamase Class A – EC: 3.5.2.6

– PDB: 1btl – Reaction:  -lactam + H2O   -amino acid – Active site residues: S70, K73, S130, E166 – Plausible mechanism: O Ser O H Lys NH 2 N H O Ser Ser O O N H O Lys NH 3 + H O Ser Glu O O H Ser O O N H Lys NH 3 + O Ser Glu OH O H O O O N H Ser H NH Lys O Ser

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

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

Pepsin

Aspartic Proteinase - Active Site residues - [DTG]x2

Eukaryotic & Fungal Aspartic Proteinases: all-atom DTG-DTG Template

Aspartic Proteases: Active Site Template

A template of 8 atoms is sufficient to identify all Aspartic Proteinases

Gly C



Asp CO 2 Gly C



Asp O



Thr/Ser O



Thr O



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

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

Database of enzyme active site templates 189 templates IIAglc histidine kinase Cholesterol oxidase GARTfase

…

Carbamoylsarcosine amidohhydrase Dihydrofolate reductase Ser-His-Asp catalytic triad

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

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) Experimentally confirmed by hydrolase assays Novel carboxylesterase acting on short acyl chain substrates

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

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

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

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

ProFunc – function from 3D structure

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

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

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