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 1) 1)Main Approaches a) a)Initial Purpose: P-Bodies b) b)Second Purpose: HIT Family c) c)Final Purpose: DcpS Family 2) 2)DcpS Family a) a)Biological Aspects b) b)Basic Analysis c) c)Extended Analysis

1 st PART: main approaches

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

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 !!! Look for the protein belonging to the family with more documented structures: HIT Family Pfam ProteinFamilyInteractionsSpeciesStructures DcpSHIT SOLUTION MAIN PROBLEM

second Purpose: hit family Histidine Triad / HIT Motif / HIT hexapeptide H  H  H   = Hydrophobic residue >swissprot|Q96C86|DCPS_HUMAN MADAAPQLGKRKRELDVEEAHAASTEEKEAGVGNGTCAPVRLPFSGFRLQKVLRE SARDK IIFLHGKVNEASGDGDGEDAVVILEKTPFQVEQVAQLLTGSPELQLQFSNDIYSTYHL FP PRQLNDVKTTVVYPATEKHLQKYLRQDLRLIRETGDDYRNITLPHLESQSLSIQWVY NIL DKKAEADRIVFENPDPSDGFVLIPDLKWNQQQLDDLYLIAICHRRGIRSLRDLTPEHL PL LRNILHQGQEAILQRYRMKGDHLRVYLHYLPSYYHLHVHFTALGFEAPGSGVERAHL LAE VIENLECDPRHYQQRTLTFALRADDPLLKLLQEAQQS

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

Mutagenesis Studies: H  H => N)  H 

Same Sequence. Different Substrates.

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

ClustalW Filter out mutated sequences Select consensus sequences T-CoffeeStampAlignfit + Stamp

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

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

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

2 nd PART: DCPS family

Biological aspects Main degradation pathway In mammals, this family contains only one member, DcpS, which: Stands for “ s cavenger mRNA d e c a p ping 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.

Biological aspects DcpS shares functional similarity with Dcp2:

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

BASIC analysis Human (Homo sapiens)Mouse (Mus musculus) Yeast (Sacch. cerevisiae) Rat (Rattus norvegicus)Bovine (Bos taurus) Pig (Sus scrofa) TARGET SEQUENCE (no PDB Structure) TEMPLATES (PDB Structure) Several orthologues have been studied:

BASIC ANALYSIS TEMPLATES (PDB Structure) 1XMM Human (Homo Sapiens) + 1VLR Mouse (Mus musculus) STAMP 86 % Sequence Identity

BASIC ANALYSIS TEMPLATES (PDB Structure) 1XMM Human (Homo Sapiens) + 1VLR Mouse (Mus musculus) 86 % Sequence Identity DALI

BASIC ANALYSIS TEMPLATES (PDB Structure) 1XMM Human (Homo Sapiens) + 1VLR Mouse (Mus musculus) 86 % Sequence Identity SAP

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 MADTAPQPSKRKRERDPEEAEAPSTEEKEARVGNGTSAPVRLPFSGFRVKKVLR ESARDK IIFLHGKVNEASGDGDGEDAIVILEKTPFQVDQVAQLLMGSPELQLQFSNDIYSTYH LFP PRQLSDVKTTVVYPATEKHLQKYLHQDLHLVRETGGDYKNITLPHLESQSLSIQWV YNIL DKKAEADRIVFENPDPSDGFVLIPDLKWNQKQLDDLYLIAICHRRGIKSLRDLTPEH LPL LRNILREGQEAILQRYQVTGDRLRVYLHYLPSYYHLHVHFTALGFEAPGAGVERAH LLAE VIENLEQDPEHYQRRTLTFALRADDPLLTLLQEAQRS

BASIC ANALYSIS TARGET DCPS_Pig ClustalWT-CoffeeStamp + DCPS Templates (2)

BASIC ANALYSIS SwissModel

BASIC ANALYSIS TARGET DCPS_Pig ClustalWT-CoffeeStamp + DCPS Templates (2)

BASIC ANALYSIS STAMP

extended analysis DcpS protein HUMAN (337 AA) DcpS N-terminal domain ( ) DcpS C-terminal domain ( ) HIT domain ( ) HIT MOTIF ( )

extended analysis DcpS dimer in complex with m 7 GpppG where: N-terminal domain – swapped dimer C-terminal domain dimer Chain A Chain B

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

extended analysis We do the analysis through 3 different approaches: SequenceStructureLiteratureBLASTDALIPubMed

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

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

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.”

extended analysis Basically members of the DcpS and HIT Families No new information related to other families DcpS C-terminal domain BLASTDALIPubMed

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

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

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

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

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

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

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

extended analysis DcpS dimer in complex with m 7 GpppG where: N-terminal domain – swapped dimer C-terminal domain dimer Chain A Chain B DcpS N-terminal domain Literature: PubMed DcpS forms swapped – HOMODIMERS !!!

extended analysis DcpS N-terminal domain Literature: PubMed “N-terminal domain shares structural homology to 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. ”

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

Thank you Any questions?