1. ©CMBI 2005 Exploring Protein Sequences - Part 2 Part 1: Patterns and Motifs Profiles Hydropathy Plots Transmembrane helices Antigenic Prediction Signal Peptides Repeats Coiled Coils Linkers Part 2: Protein Domains Domain databases Celia van Gelder CMBI Radboud University December 2005

2. ©CMBI 2005 Definition of protein domains Group of residues with high contact density, number of contacts within domains is higher than the number of contacts between domains. A stable unit of protein structure that can fold autonomously A rigid body linked to other domains by flexible linkers A portion of the protein that can be active on its own if you remove it from the rest of the protein.

3. ©CMBI 2005 Protein Domains Domains can be 25 to 500 residues long; most are less than 200 residues The average protein contains 2 or 3 domains The total number of different types of domains ~1000 – 3000 The same or similar domains are found in different proteins. “Nature is a ‘tinkerer’ and not an inventor” (Jacob, 1977). “Nature is smart but lazy” Usually, each domain plays a specific role in the function of the protein.

4. ©CMBI 2005

5. Linkers Domain linkers link the protein domains together and have been found to contain an amino acid signature that is distinct from the structurally compact domains. Average linker size 8-9 amino acids Linkers are susceptible for protease attack and they are flexible.

6. ©CMBI 2005 Protein Domain Databases Even though the structure of a domain is not always known it is still possible to define the domain boundaries from sequence alone Many of the common domains have already been defined in domain databases Advantages: Pre-annotated domains Easy interpretation of domain structure Problem: Not trivial to define domain boundaries unambiguously

7. Protein Domains http://ip30.eti.uva.nl/ember-demo/ch3

8. ©CMBI 2005 Domain databases (2) Generation#entries PfamAmanual7503 families PfamBautomatic>140,000 families Printsmanual11,170 motifs Prosite Profilesmanual577 profiles Blocksautomatic28,337 blocks, 5733 groups SMARTmanual667 HMMs ProDomautomatic501,917 domain families

9. ©CMBI 2005 PRINTS database Most protein families are characterised not by one, but by several conserved motifs Fingerprints are groups of conserved motifs excised from sequence alignments Taken together, they provide diagnostic family signatures. They are are the basis of the PRINTS database, and are stored in the form of aligned motifs Input about protein families is done manually True members match all elements of the fingerprint in order, subfamily members may match part of fingerprint

10. PRINTS database http://ip30.eti.uva.nl/ember-demo/ch3

11. ©CMBI 2005 PRINTS

12. ©CMBI 2005 BLOCKS database Blocks are multiply aligned ungapped segments corresponding to the most highly conserved regions of proteins. The blocks for the BLOCKs database are made automatically by looking for the most highly conserved regions in groups of proteins documented in InterPro. Version 14.1 of the BLOCKS Database consists of 28,337 blocks representing 5733 groups documented in InterPro 8.1 (february 2005) To ensure complete coverage it is recommended that both the PRINTS and the BLOCKS database be searched

13. ©CMBI 2005

15. Typical sequence logo illustrating three highly conserved positions within a 14-residue motif. The motif runs along the x-axis, while the y- axis denotes the information content for each residue at each position. This logo shows 3 well-conserved positions (marked with a *), of which only the last is totally conserved. The colours broadly indicate residue properties - red for acidic, blue for basic, etc. http://ip30.eti.uva.nl/ember-demo/

16. ©CMBI 2005 ProDom: The Protein Domain Database ProDom is a comprehensive set of protein domain families automatically generated Each entry provides a multiple sequence alignment of homologous domains and a family consensus sequence. Current ProDom release: ProDom 2004.1, June 2004, 501917 domain families

17. ©CMBI 2005

19. Pfam Pfam (Protein families) is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. For each family in Pfam you can: Look at multiple alignments View the domain organisation of proteins Examine species distribution Follow links to other databases View known protein structures

20. ©CMBI 2005 Pfam Two distinct parts: –Pfam-A entries are manually curated 7503 families –Pfam-B entries automatically generated clusters >140,000 (not covered by Pfam-A) New: iPfam is a resource that describes domain-domain interactions that are observed in known structures

21. ©CMBI 2005

23. SMART SMART - Simple Modular Architecture Research Tool Domain families found in: 1) signalling 2) nuclear 3) extracellular 4) other Current version 5.0: Number of SMART HMMs: 669 You can use SMART in two different modes: normal or genomic.

24. ©CMBI 2005 Bacteriorhodopsin Human serine protease

25. ©CMBI 2005 Additional features of SMART Used for identification of genetically mobile domains and analysis of domain architectures Can search for proteins containing specific combinations of domains in defined taxa Can search for proteins with identical domain architecture Also has information on intrinsic features like signal sequences, transmembrane helices, coiled-coil regions and compositionally biased regions

26. ©CMBI 2005 Limitations of domain databases Patterns not present for all families of proteins Multiple sequence alignment to define patterns could be inaccurate due to an automatic alignment Low number of sequences from different species could result in inaccurate patterns

27. ©CMBI 2005 Integrating Pattern databases InterPro - Integrated Documentation Resource of Protein Families, Domains and Functional Sites. InterPro is a database of protein families, domains and functional sites in which identifiable features found in known proteins can be applied to unknown protein sequences. The aim is to provide a one-stop-shop for protein family diagnostics

28. ©CMBI 2005 InterPro Member Databases Prosite (regular expressions and profiles) Pfam, SMART, TIGRFAMs, PIRSF, PANTHER, Gene3D and SUPERFAMILY (hidden Markov Models - HMMs) PRINTS (groups of aligned, un-weighted motifs) ProDom (uses cluster analysis to group sequences) Release 12.0 contains 12542 entries Types of entries: Family, Domain, Repeat, PTM, Binding Site, Active Site

29. ©CMBI 2005

32. Summary Many different protein signature databases exist (from small patterns to alignments to complex HMMs) The databases have different strengths and weaknesses. Some databases can be better for your sequence than others Therefore: best to combine methods, preferably in an integrated database The quality of a database/server is best tested with a sequence you know very well Always do control experiments: never trust a server