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Study of the DcpS Family

Study of the DcpS Family. March 5 th 2009 Structural Bioinformatics Msc BIOINFO, UPF. Salvador Jesús Capella Gutiérrez Juan Ramón Meneu Hernández Rut Carolina Morata Gil. Main Scheme. Main Approaches Initial Purpose: P-Bodies Second Purpose: HIT Family Final Purpose: DcpS Family

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Study of the DcpS Family

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  1. Study of theDcpS Family March 5th 2009 Structural Bioinformatics Msc BIOINFO, UPF Salvador Jesús Capella Gutiérrez Juan Ramón Meneu Hernández Rut Carolina Morata Gil

  2. Main Scheme • Main Approaches • Initial Purpose: P-Bodies • Second Purpose: HIT Family • Final Purpose: DcpS Family • DcpS Family • Biological Aspects • Basic Analysis • Extended Analysis

  3. 1st PART: main approaches

  4. Initial Purpose: P-Bodies P-Bodies are discrete cytoplasmic domains where several proteins involved in mRNA degradation, translational repression and some other related functions colocalize

  5. Initial Purpose: P-Bodies P-Bodies are discrete cytoplasmic domains where several proteins involved in mRNA degradation, translational repression and some other related functions colocalize Constituted by proteins belonging to different families !!! MAIN PROBLEM Look for the protein belonging to the family with more documented structures: HIT Family SOLUTION Pfam

  6. second Purpose: hit family Histidine Triad / HIT Motif / HIT hexapeptide H j H j H j j= Hydrophobic residue >swissprot|Q96C86|DCPS_HUMAN MADAAPQLGKRKRELDVEEAHAASTEEKEAGVGNGTCAPVRLPFSGFRLQKVLRESARDK IIFLHGKVNEASGDGDGEDAVVILEKTPFQVEQVAQLLTGSPELQLQFSNDIYSTYHLFP PRQLNDVKTTVVYPATEKHLQKYLRQDLRLIRETGDDYRNITLPHLESQSLSIQWVYNIL DKKAEADRIVFENPDPSDGFVLIPDLKWNQQQLDDLYLIAICHRRGIRSLRDLTPEHLPL LRNILHQGQEAILQRYRMKGDHLRVYLHYLPSYYHLHVHFTALGFEAPGSGVERAHLLAE VIENLECDPRHYQQRTLTFALRADDPLLKLLQEAQQS

  7. second Purpose: hit family TARGET DCPS_Human HMMPfam against PFAM Blast against Uniref100 DCPS Family HMMFetch against PFAM PSSM Matrix + target + target HMM Matrix Blast against PDB HMMSearch against PDB results Filter out mutated sequences Select consensus sequences

  8. MutagenesisStudies: H j (H => N) j H j

  9. Same Sequence. Different Substrates.

  10. Same Sequence. Different Substrates.

  11. Same Sequence. Different Substrates.

  12. second Purpose: hit family 9 final structures for the HIT Family

  13. second Purpose: hit family Filter out mutated sequences Select consensus sequences ClustalW T-Coffee Stamp Alignfit + Stamp

  14. second Purpose: hit family ALIGNFIT + STAMP STAMP ALIGNFIT + STAMP STAMP 9 final structures for the HIT Family Superposition turned out to be a real mess !!!

  15. second Purpose: hit family Histidine Triad / HIT Motif / HIT hexapeptide H j H j H j j= Hydrophobic residue HIT Superfamily DcpS Fhit Hint Within each branch => High degree of conservation among proteins Between each branch => HIT MOTIF is the only region absolutely conserved

  16. final Purpose: dcps family DcpS Family 9 final structures for the HIT Family Superposition turned out to be a real mess !!!

  17. 2nd PART: DCPS family

  18. Biological aspects In mammals, this family contains only one member, DcpS, which: Stands for “scavenger mRNA decapping enzyme” Hydrolyses the residual cap structure following 3' to 5' mRNA decay Is the first member of the HIT family of proteins with a defined biological function. Main degradation pathway

  19. Biological aspects DcpS shares functional similarity with Dcp2:

  20. Biological aspects Mutations in the HIT motif lead to the complete loss of the function The region (binding site) is critical for decapping activity

  21. BASIC analysis Several orthologues have been studied: TEMPLATES (PDB Structure) TARGET SEQUENCE (no PDB Structure)

  22. BASIC ANALYSIS TEMPLATES (PDB Structure) 86 % Sequence Identity + STAMP

  23. BASIC ANALYSIS TEMPLATES (PDB Structure) 86 % Sequence Identity + DALI

  24. BASIC ANALYSIS TEMPLATES (PDB Structure) 86 % Sequence Identity + SAP

  25. BASIC ANALYSIS TARGET SEQUENCE (no PDB Structure) >sp|Q8MIZ3|DCPS_PIG Scavenger mRNA-decapping enzyme DcpS OS=Sus scrofa GN=DCPS PE=2 SV=1 MADTAPQPSKRKRERDPEEAEAPSTEEKEARVGNGTSAPVRLPFSGFRVKKVLRESARDK IIFLHGKVNEASGDGDGEDAIVILEKTPFQVDQVAQLLMGSPELQLQFSNDIYSTYHLFP PRQLSDVKTTVVYPATEKHLQKYLHQDLHLVRETGGDYKNITLPHLESQSLSIQWVYNIL DKKAEADRIVFENPDPSDGFVLIPDLKWNQKQLDDLYLIAICHRRGIKSLRDLTPEHLPL LRNILREGQEAILQRYQVTGDRLRVYLHYLPSYYHLHVHFTALGFEAPGAGVERAHLLAE VIENLEQDPEHYQRRTLTFALRADDPLLTLLQEAQRS

  26. BASIC ANALYSIS TARGET DCPS_Pig + DCPS Templates (2) ClustalW T-Coffee Stamp

  27. BASIC ANALYSIS SwissModel

  28. BASIC ANALYSIS TARGET DCPS_Pig + DCPS Templates (2) ClustalW T-Coffee Stamp

  29. BASIC ANALYSIS STAMP STAMP

  30. extended analysis DcpS protein HUMAN (337 AA) DcpS N-terminal domain (40 - 145) DcpS C-terminal domain (146 - 337) HIT MOTIF (275 - 279) HIT domain (268 - 279)

  31. extended analysis N-terminal domain – swapped dimer C-terminal domain dimer DcpS dimer in complex with m7GpppG where: Chain A Chain B

  32. extended analysis After analysing the HIT domain, we go deep into… DcpS N-terminal domain DcpS C-terminal domain … to look for hints on the specificity of DcpS >swissprot|Q96C86|DCPS_HUMAN Scavenger mRNA-decapping enzyme DcpS; MADAAPQLGKRKRELDVEEAHAASTEEKEAGVGNGTCAPVRLPFSGFRLQKVLRESARDK IIFLHGKVNEASGDGDGEDAVVILEKTPFQVEQVAQLLTGSPELQLQFSNDIYSTYHLFP PRQLNDVKTTVVYPATEKHLQKYLRQDLRLIRETGDDYRNITLPHLESQSLSIQWVYNIL DKKAEADRIVFENPDPSDGFVLIPDLKWNQQQLDDLYLIAICHRRGIRSLRDLTPEHLPL LRNILHQGQEAILQRYRMKGDHLRVYLHYLPSYYHLHVHFTALGFEAPGSGVERAHLLAE VIENLECDPRHYQQRTLTFALRADDPLLKLLQEAQQS

  33. extended analysis We do the analysis through 3 different approaches: Literature Sequence Structure BLAST DALI PubMed

  34. extended analysis DcpS C-terminal domain Sequence: BLAST Members of the DcpS Family Predicted or putative proteins (not reviewed) No new information related to other families

  35. extended analysis DcpS C-terminal domain Structure: DALI All the results are members of the DcpS and HIT Families No new information related to other families

  36. extended analysis DcpS C-terminal domain Literature: PubMed The C-Terminal DcpS Domain Is Related, but Distinct from the HIT Protein Family “A DALI search of the Protein Data Bank revealed structural similarity between DcpS and a number of HIT proteins. ...” “… . In addition and as noted above, the DcpS C-terminal domain is not sufficient for cap hydrolysis, indicating substantive differences between these protein families.”

  37. extended analysis DcpS C-terminal domain Basically members of the DcpS and HIT Families BLAST DALI PubMed No new information related to other families

  38. extended analysis DcpS N-terminal domain Sequence: BLAST Members of the DcpS Family Predicted or putative proteins (not reviewed) No new information related to other families

  39. extended analysis DcpS N-terminal domain Literature: PubMed PDB search using DALI N-terminal domain shares structural homologyto NTF2-like proteins Carotenoid binding protein (CBP) Yeast mRNA export factor Mex67 Metazoan mRNA export factor p15

  40. extended analysis DcpS N-terminal domain Literature: PubMed DcpS DALI

  41. extended analysis DcpS N-terminal domain Literature: PubMed DcpS mRNA export factor p15 DALI

  42. extended analysis DcpS N-terminal domain Literature: PubMed DcpS mRNA export factor p15 Carotenoid binding prot (CBP) DALI

  43. extended analysis DcpS N-terminal domain Literature: PubMed DcpS mRNA export factor p15 Carotenoid binding prot (CBP) mRNA export factor Mex67 DALI

  44. extended analysis DcpS N-terminal domain Literature: PubMed These NTF2-like proteins form HETERODIMERS

  45. extended analysis DcpS N-terminal domain Literature: PubMed DcpS forms swapped – HOMODIMERS !!! N-terminal domain – swapped dimer C-terminal domain dimer DcpS dimer in complex with m7GpppG where: Chain A Chain B

  46. extended analysis DcpS N-terminal domain Literature: PubMed “N-terminal domain shares structural homologyto NTF2-like proteins …” BUT “… DcpS domain swapped – dimer has a unique topology and organization, which is different from either Mex67/Mtr2 or p15/TAP complexes “Mex67/Mtr2 and p15/TAP have been implicated in mRNA export pathways …” BUT “… Any functional significance to the DcpS N-terminal domain structure remains unclear. ”

  47. extended analysis DcpS N-terminal domain Structure: DALI A PDB search using DALI shows kind of structural homology with proteins belonging to some other families Further studies should be carried out

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