Study of theStudy of the
DcpS FamilyDcpS FamilyMarch 5th 2009
Structural Bioinformatics
Msc BIOINFO, UPF
Salvador Jesús Capella Gutiérrez
Juan Ramón Meneu Hernández
Rut Carolina Morata Gil
Main SchemeMain Scheme
1) Main Approachesa) Initial Purpose: P-Bodiesb) Second Purpose: HIT Familyc) Final Purpose: DcpS Family
2) DcpS Familya) Biological Aspectsb) Basic Analysisc) Extended Analysis
11stst PART: PART:
main approachesmain approaches
Initial Purpose: P-BodiesInitial 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-BodiesInitial 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
Protein Family Interactions
Species Structures
DcpS HIT 1 932 33
SOLUTION
MAIN PROBLEM
second Purpose: hit familysecond Purpose: hit family
Histidine Triad / HIT Motif / HIT hexapeptide
H H H = Hydrophobic residue
>swissprot|Q96C86|DCPS_HUMANMADAAPQLGKRKRELDVEEAHAASTEEKEAGVGNGTCAPVRLPFSGFRLQKVLRESARDKIIFLHGKVNEASGDGDGEDAVVILEKTPFQVEQVAQLLTGSPELQLQFSNDIYSTYHLFPPRQLNDVKTTVVYPATEKHLQKYLRQDLRLIRETGDDYRNITLPHLESQSLSIQWVYNILDKKAEADRIVFENPDPSDGFVLIPDLKWNQQQLDDLYLIAICHRRGIRSLRDLTPEHLPLLRNILHQGQEAILQRYRMKGDHLRVYLHYLPSYYHLHVHFTALGFEAPGSGVERAHLLAEVIENLECDPRHYQQRTLTFALRADDPLLKLLQEAQQS
results
PSSM Matrix
second Purpose: hit familysecond 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.
Same Sequence. Different Substrates.
Same Sequence. Different Substrates.
9 final structures for the HIT Family
second Purpose: hit familysecond Purpose: hit family
second Purpose: hit familysecond Purpose: hit family
ClustalW
Filter out mutated sequences
Select consensus sequences
T-Coffee StampAlignfit +
Stamp
second Purpose: hit familysecond Purpose: hit family
9 final structures for the HIT
Family
Superposition turned out to be a real mess
!!!
ALIGNFIT + STAMPSTAMPSTAMPALIGNFIT + STAMP
second Purpose: hit familysecond 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 familyfinal Purpose: dcps family
22ndnd PART: PART:
DCPS familyDCPS family
Biological aspectsBiological aspects
Main degradation pathway
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.
Biological aspectsBiological aspects
DcpS shares functional similarity with Dcp2:
Biological aspectsBiological aspects
Mutations in the HIT motif lead to the complete loss of
the function
The region (binding site) is critical for decapping activity
BASIC analysisBASIC 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 ANALYSISBASIC ANALYSIS
TEMPLATES (PDB Structure)
1XMM
Human (Homo Sapiens)
+
1VLR
Mouse (Mus musculus)
STAMP
86 % Sequence Identity
BASIC ANALYSISBASIC ANALYSIS
TEMPLATES (PDB Structure)
1XMM
Human (Homo Sapiens)
+
1VLR
Mouse (Mus musculus)
86 % Sequence Identity
DALI
BASIC ANALYSISBASIC ANALYSIS
TEMPLATES (PDB Structure)
1XMM
Human (Homo Sapiens)
+
1VLR
Mouse (Mus musculus)
86 % Sequence Identity
SAP
BASIC ANALYSISBASIC ANALYSIS
TARGET SEQUENCE (no PDB Structure)
>sp|Q8MIZ3|DCPS_PIG Scavenger mRNA-decapping enzyme DcpS OS=Sus scrofa GN=DCPS PE=2 SV=1MADTAPQPSKRKRERDPEEAEAPSTEEKEARVGNGTSAPVRLPFSGFRVKKVLRESARDKIIFLHGKVNEASGDGDGEDAIVILEKTPFQVDQVAQLLMGSPELQLQFSNDIYSTYHLFPPRQLSDVKTTVVYPATEKHLQKYLHQDLHLVRETGGDYKNITLPHLESQSLSIQWVYNILDKKAEADRIVFENPDPSDGFVLIPDLKWNQKQLDDLYLIAICHRRGIKSLRDLTPEHLPLLRNILREGQEAILQRYQVTGDRLRVYLHYLPSYYHLHVHFTALGFEAPGAGVERAHLLAEVIENLEQDPEHYQRRTLTFALRADDPLLTLLQEAQRS
BASIC ANALYSISBASIC ANALYSIS
TARGET DCPS_Pig
ClustalW T-Coffee Stamp
+ DCPS Templates (2)
BASIC ANALYSISBASIC ANALYSIS
SwissModel
BASIC ANALYSISBASIC ANALYSIS
TARGET DCPS_Pig
ClustalW T-Coffee Stamp
+ DCPS Templates (2)
BASIC ANALYSISBASIC ANALYSIS
STAMPSTAMP
extended analysisextended analysis
DcpS protein HUMAN (337 AA)
DcpS N-terminal domain(40 - 145)
DcpS C-terminal domain(146 - 337)
HIT domain(268 - 279)
HIT MOTIF(275 - 279)
extended analysisextended analysis
DcpS dimer in complex with m7GpppG where:
N-terminaldomain – swapped
dimer
C-terminaldomain dimer
Chain AChain B
extended analysisextended 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;MADAAPQLGKRKRELDVEEAHAASTEEKEAGVGNGTCAPVRLPFSGFRLQKVLRESARDKIIFLHGKVNEASGDGDGEDAVVILEKTPFQVEQVAQLLTGSPELQLQFSNDIYSTYHLFPPRQLNDVKTTVVYPATEKHLQKYLRQDLRLIRETGDDYRNITLPHLESQSLSIQWVYNILDKKAEADRIVFENPDPSDGFVLIPDLKWNQQQLDDLYLIAICHRRGIRSLRDLTPEHLPLLRNILHQGQEAILQRYRMKGDHLRVYLHYLPSYYHLHVHFTALGFEAPGSGVERAHLLAEVIENLECDPRHYQQRTLTFALRADDPLLKLLQEAQQS
… to look for hints on the specificity of DcpS
extended analysisextended analysis
We do the analysis through 3 different approaches:
Sequence
Structure
Literature
BLAST DALIPubMed
extended analysisextended 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 analysisextended 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 analysisextended 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 analysisextended analysis
Basically members of the
DcpS and HIT Families
No new information related to other families
DcpS C-terminal domain
extended analysisextended analysis
Members of the DcpS FamilyPredicted or putative proteins
(not reviewed)
No new information
related to other families
DcpS N-terminal domain
Sequence: BLAST
extended analysisextended 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 analysisextended analysis
DcpS N-terminal domain
Literature: PubMed
DcpS
DALI
extended analysisextended analysis
DcpS N-terminal domain
Literature: PubMed
DcpS
mRNA export factor p15
DALI
extended analysisextended analysis
DcpS N-terminal domain
Literature: PubMed
DcpS
mRNA export factor p15Carotenoid binding prot (CBP)
DALI
extended analysisextended analysis
DcpS N-terminal domain
Literature: PubMed
DcpS
mRNA export factor p15Carotenoid binding prot (CBP)mRNA export factor Mex67
DALI
extended analysisextended analysis
DcpS N-terminal domain
Literature: PubMed
These NTF2-like proteins form HETERODIMERS
extended analysisextended analysis
DcpS dimer in complex with m7GpppG where:
N-terminaldomain – swapped
dimer
C-terminaldomain dimer
Chain AChain B
DcpS N-terminal domain
Literature: PubMed
DcpS forms swapped – HOMODIMERS !!!
extended analysisextended 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 analysisextended 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 youThank you
Any questions?Any questions?