Clustering and Motif Discovery
in Kinases of Yeast, Worm and
Arabidopsis thaliana
Sihui Zhao
Background – Kinase
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Protein kinases play a pivotal role in
the control of all cellular processes
Cell proliferation, differentiation,
adhesion, migration, metabolism and
signal transduction
A kinase superfamily in each genome,
~2% of all sequences
Background
Structure of Catalytic Domain
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Also called C-subunit
Conserved among protein kinase
superfamily
Contains 250-300 residues
12 subdomains
Background
Subdomains of C-subunit
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Two pivital subdomains (based on
PKA):
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Subdomain I: Sequester ATP
Gly-X-Gly-X-X-Gly-X-Val
Subdomain VIB: ‘Catalytic loop’
His-Arg-Asp-X-Lys-X-X-Asn
Background
Conserved Residues
Residue
Probable Function
Gly50 Gly52 Val57
Sequester ATP
Lys72 Glu91
Positioning triphosphate group
Asp166 Lys168 Asn171
Catalytic loop
Glu208 Arg280
Assembly of catalytic core
Asp220
Assembly of catalytic loop
Background
Motif
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Motif is a locally conserved region
Conserved due to higher selection
pressure compared to non-conserved
regions
Importance to the biological function or
structure
Problem & Strategy
in Motif Discovery
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Background
Motif discovery relies on either statistical
or combinatorial pattern search
techqniues
Problem: High noise compared to signal
when facing huge number of sequences
Strategy: Clustering/classification used
to find sequence families first to
decrease the noise ratio
Objectives
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Cluster kinase sequences into different
families
Find conserved motifs from sequence
families
Tools
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Blast – Sequence alignment tool
ClustalW – Multiple alignment tool
HMMER – HMM-based package
BAG package – Sequence clustering
package
BlockerMaker – Block/Motif discovery tool
LAMA – Alignment tool for Blocks
Perl
Computational Framework
– Outline
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Collecting and clustering kinase
sequences based on similarity
The iterative HMM search – To collect
more kinases, especially remotely
homologous sequences
Motif discovery – To find blocks from
each cluster and merge blocks across
multiple clusters
Computational Framework
Collecting and Clustering Sequences
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Cluster kinase sequences
Extract annotated kinase sequences
All to all pairwise comparison
Estimate best score for clustering
Cluster sequences using BAG
Computational Framework
HMM Iterative Search
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Collect more sequences for each cluster
Multiple alignment using CLUSTALW
Build HMM/Profile
Search all 3 genomes
Add hits to each cluster if any
Computational Framework
Motif Discovery
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Find blocks and merge across multiple clusters
Block discovery by BlockMaker
All to all block comparison by LAMA
Clustering blocks using BAG package
Conserved sites detection
Result
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963 kinase from ~45,000 sequences
(~2%)
159 clusters of kinase sequences
containing 2 to 32 sequences each
0 to ~1000 sequences added to each
cluster after HMM iterative search
Result
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71 sequence clusters sent to BlockMaker
ID
c51.seq-1 BLOCK
AC
c51.seq-1; distance from previous block=(79,120)
DE
similar to eukaryotic protein kinase domains
BL
EGL motif=[5,0,17] motomat=[1,1,-10] width=31 seqs=5
gi|3329644|gb|AAC
( 792) SNFNFEFHKDSLEILEPIGSGHFGVVRRGIL
99
gi|3329650|gb|AAC
( 154) YNPKYEVDLEKLEILEQLGDGQFGLVNRGLL
92
gi|3877967|emb|CA
( 836) YNNDYEIDPVNLEILNPIGSGHFGVVKKGLL
79
gi|3877968|emb|CA
( 842) YNEDYEIDLENLEILETLGSGQFGIVKKGYL
77
gi|3878749|emb|CA
( 129) YKKQYEIASENLENKSILGSGNFGVVRKGIL 100
Result
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45 clusters of Blocks after LAMA
comparison and BAG clustering
Result
Some Found Conserved Sites
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Cluster 11, size 29
Subdomain I: G-X-G-X-X-G-X-V
Cluster 16, size 97
Subdomain VIB: H-R-D-X-K-X-X-N
Result
Some New Sites
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Cluster 20, size=8
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Known: Arg280 - assembly of catalytic core
Unknown: Cys, Trp, Pro
Cluster 31, size=13 Alignment and motif
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Alignment and motif
Known: Asp220 - assembly of catalytic loop
Unknown: Gly, Thr, Tyr
Cluster 40, size=7
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Alignment and motif
Known: Glu91 - positioning triphosphate group
Unknown: His, Pro
Conclusion
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This computational framework is
successful
Especially when no preliminary
information on huge amount of
sequences
It’s efficient
Not completely automatic
Conclusion
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Kinases are clustered based on
similarity, which provides a way to
deduce the functions from other family
members
Some new conserved sites are found,
which might indicate the specificity of
kinase functions
Acknowledgement
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Prof. Sun Kim
Prof. Mehmet Dalkilic
Dr. Irfan Gunduz