Download Evolution, structure and function of YlqF

Evolution, structure and
function of 1pujA
Scott L. Allen, Alexander Mulherin, Takayuki Hasegawa
Introduction
• 1pujA is a known GTPase of Bacillus
subtilis (Matsuo et. al. 2006) which is of
the YlqF family of proteins (Leipe, D. et. al.
2002).
• YlqF proteins are a subfamily of the P-loop
NTPases superfamily.
• YlqF are Characterised by a circular
permutation (Leipe, D. et. al. 2002)
Introduction
• P-loop NTPases are one of the most
abundant gene products.
• Can compromise up to 20% of an
organisms proteome.
• Known to regulate many fundamental
cellular processes such as translation,
cell-signalling, intracellular trafficking and
cytoskeletal re-organisation (Anand, B. et.
al. 2006; Leipe, D. et. al. 2002).
Introduction
• YlqF of Bacillus subtilis has previously
been associated with the assembly of the
50S ribosomal subunit.
• Cells in which YlqF was inhibited showed
slow growth and a build up of mis-folded
50S ribosomal subunits (Matsuo, Y. et. al.
2006).
Introduction
• Computational biological methods
incorporating, functional, structural and
phylogenetic analyses will be used to Infer
the function of 1pujA and related YlqF
proteins.
FUNCTIONAL ANALYSIS
Literature Search:
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Google scholar, PubMed
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Matsuo et al. and Uicker et al. who both reached the conclusion that YlqF
played a major role as a GTPase in 50s ribosomal subunit formation.
ProFunc:
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Most commonly occurring name term: GTPase, followed by GTP
binding.
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ProFunc Results: Interpro, PDB, SSM and DALI and 3D functional
template all significant however UniProt was the most promising.
UniProt:
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Aligned sequences:
%-tage
Seq. id. identity Name
-----------------Query
- Query sequence
O31743 95.6 - O31743 YlqF protein.
Q65JP4 84.6 - Q65JP4 YlqF (GTP-binding domain protein).
Q845L2 70.1 - Q845L2 Hypothetical protein.
Q9Z9S1 59.9 - Q9Z9S1 YlqF (BH2476 protein).
Q4MFY1 65.9 - Q4MFY1 Hypothetical protein.
Q5WFP0 53.5 - Q5WFP0 GTPase.
Q5HPU7 50.9 - Q5HPU7 GTP-binding protein, putative.
Q3XZB6 52.4 - Q3XZB6 GTP-binding protein.
A0Q0X8 49.3 - A0Q0X8 GTP-binding protein, putative.
Q03KX8 47.6 - Q03KX8 Predicted GTPase.
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A multiple sequence alignment search returned 10 significant matches 2
percentage identities higher then 80%.
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All of the proteins are either GTPases or GTP-binding proteins.
SymAtlas:
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Relatively conserved throughout.
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Consistent with a protein essential for fundamental biological processes
ProKnow:
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GO Code.
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The results indicate that GTP binding is the molecular function followed by
nucleotide binding, methyltransferase activity and GTPase activity.
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The biological process is small GTPase mediated signal transduction, which
refers to any series of molecular signals in which a small monomeric
GTPase relays one or more of the signals
0005525
0000166
0008168
0003924
0007264
Bayesian Score. Evidence Rank. Number of Clues. Description
Molecular Function
0.9815
2.8
6
GTP binding
0.0101
2.9
6
nucleotide binding
0.0068
2.9
6
methyltransferase activity
0.0016
2.5
6
GTPase activity
Biological Process
1.0000
2.8
6
small GTPase mediated signal transduction
Sturucture
Overall Structure
• 282 amino acids
• 2 ligands – magnesium ion &
phosphoaminophosphonic acid-guanylate
ester
• The molecular weight: 31986 Da
• The secondary structure: 50 % alpha
helices & 10% beta sheets
The sequence of YlqF
• MTIQWFPGHMAKARREVTEKLKLIDIVYELVDARIPMSS
RNPMIEDILKNKPRIMLLNKADKADAAVTQQ
• WKEHFENQGIRSLSINSVNGQGLNQIVPASKEILQEKF
DRMRAKGVKPRAIRALIIGIPNVGKSTLINRL
• AKKNIAKTGDRPGITTSQQWVKVGKELELLDTPGILWP
KFEDELVGLRLAVTGAIKDSIINLQDVAVFGL
• RFLEEHYPERLKERYGLDEIPEDIAELFDAIGEKRGCLM
SGGLINYDKTTEVIIRDIRTEKFGRLSFEQP
• TM
The image of YlpF
• The image was generated by PyMol
Sturucture Analysis
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SCOP Classification
Domain Info: d1puja_
Class: Alpha and beta proteins
Fold: P-loop containing nucleotide triphosphate
hydrolases
Superfamily: P-loop containing nucleotide
triphosphate
Family: G proteins
Domain: Probable GTPase YlqF
Species: Bacillus subtilis
Structure Analysis
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CATH Classification
Domain: 1pujA01
Class: Alpha/Beta
Architecture: 3-layer(alpha-beta-alpha)
Sandwich
• Topology: Rossmann fold
• Homology: P-loop containing nucleotide
triphosphate hydrolases
Structure Analysis
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CATH Classification
Domain: 1pujA02
Class: Mainly alpha
Architecture: Orthogonal Bundle
Topology: Conserved hypothetical protein
YlqF; Chain A; domain 2
InterProScan Results
• InterPro: IPR005289 MG442
• This is a GTP binding domain. This was found in
many different families including Ras GTPase
superfamily, HSR1-related GTP binding protein.
And they all play different functions.
• InterPro: IPR002917 MMR_HSR1
• HSR1 is placed to the human MHC class I
region. It is known to be highly homologous to a
putative GTP binding protein like MMR1 from
mouse. These are also known to represent a
new subfamily of GTP binding proteins of
prokaryote and eukaryote members.
EVOLUTION
Phylogeny
• 1pujA FASTA sequence was used as a query to BLASTP
the NR database.
• Sequence matches were selected for MSA on the basis
of presence in model organisms representative of the 3
major kingdoms of life.
• Bacteria
• Archaea
• & Eukaryotes
• MSA were performed using CLUSTAL X
• Phylogenetic tree constructed using the PHYLIP
package.
• Distance based methods.
• 100 bootstraps to determine branch confidence.
Phylogeny
• MSA
showed high
conservation
of essential
GTPase
domains
across all 3
kingdoms.
Phylogeny
• MSA identified
that 1pujA and
related proteins
as having high
conservation of
the circular
permutated
NKxD motif.
Phylogeny
• Tree
displayed
three
distinct
groupings.
Phylogeny
• A bacterial
group (blue),
an entirely
eukaryotic
group (green)
and an
archeoeukaryotic
group (red).
• Consistant
with a protein
that existed in
LUCA (Leipe,
D. 2002)
Phylogeny
• Further analysis of the MSA revealed the
the two eukaryotic groupings were due to
the archeo-eukaryotic group containing an
addition N-terminal domain.
Phylogeny
• A previous study identified this region as a
coiled coil domain.
• Pink boxes show conserved residues that
are part of the domain once folded into its
final structure.
Phylogeny
• Furthermore, this coiled coil domain is
characteristic of YawG proteins.
• Associated with RNA binding
Phylogeny
• 1pujA is a member of the YlqF family of
proteins
• High conservation of essential GTPase
motifs including the circularly permutated
NKxD motif
• YlqF and YwaG proteins are closely
related dispite YwaG’s addition coiled coil
domain
Phylogeny
• Inferred
from tree
that YlqF is
ancestral to
YawG which
may have
arisen via
domain
fusion
(Leipe, D.
et. al. 2002).
Phylogeny
• Red stars denote
less than 95%
confidence in
branch
• Still believed tree
is representative
of the true
phylogeny
• NJ trees are
based on
distance matrix
methods which
are suited for
lineages with
large variation in
the evolutionay
rate (Saitou and
Nei, 1987)
Phylogeny
• Due to the close evolutionary relationship between YlqF
and YawG proteins
• High conservation of primary structure (MSA)
• Concluded from the phylogenetic analyses that YlqF and
YawG are likely to be homologous proteins sharing
GTPase activity
• Dispite YlqF lacking the coiled coil domain of YwaG, the
high conservation of the remaining sequence could still
be suggestive that YlqF also binds RNA.
• May have evolved as an adaptation from the need for
crucial proteins to use a more stable substrate such as
GTP over ATP (Leipe, D. et. al. 2002).
Conclusion
• Structural, functional and evolutionary analyses collectively indicate
that 1pujA is a GTPase of the YlqF family.
• High conservation of YlqF over large spans of evolutionary time
indicate strong stabilising selection which suggests a role in one or
several crucial biological processes (Leipe, D. et. al. 2002).
• This is supported by the functional results which indicate YlqF has a
very important function, this is due to their level of expression in all
tissues of humans and mice.
• Although the current study can not directly show association of
1pujA with ribosomal assembly, it is concluded that YlqF proteins are
GTPase likely to be involved in a fundamental biological process
such as translation. This study support of the work by Matsuo et. al.
(2002) in which it was hypothesised that YlqF is needed for correct
assembly of the 50S ribosomal subunit.