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:

• Google scholar, PubMed

• 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:

• Most commonly occurring name term: GTPase, followed by GTP binding.

• ProFunc Results: Interpro, PDB, SSM and DALI and 3D functional template all significant however UniProt was the most promising.

UniProt:• 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.

• A multiple sequence alignment search returned 10 significant matches 2 percentage identities higher then 80%.

• All of the proteins are either GTPases or GTP-binding proteins.

SymAtlas:

• Relatively conserved throughout.

• Consistent with a protein essential for fundamental biological processes

ProKnow:• GO Code. Bayesian Score. Evidence Rank. Number of Clues. Description • Molecular Function• 0005525 0.9815 2.8 6 GTP binding• 0000166 0.0101 2.9 6 nucleotide binding• 0008168 0.0068 2.9 6 methyltransferase activity• 0003924 0.0016 2.5 6 GTPase activity• Biological Process• 0007264 1.0000 2.8 6 small GTPase mediated signal transduction

• The results indicate that GTP binding is the molecular function followed by nucleotide binding, methyltransferase activity and GTPase activity.

• 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

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

• MTIQWFPGHMAKARREVTEKLKLIDIVYELVDARIPMSSRNPMIEDILKNKPRIMLLNKADKADAAVTQQ

• WKEHFENQGIRSLSINSVNGQGLNQIVPASKEILQEKFDRMRAKGVKPRAIRALIIGIPNVGKSTLINRL

• AKKNIAKTGDRPGITTSQQWVKVGKELELLDTPGILWPKFEDELVGLRLAVTGAIKDSIINLQDVAVFGL

• RFLEEHYPERLKERYGLDEIPEDIAELFDAIGEKRGCLMSGGLINYDKTTEVIIRDIRTEKFGRLSFEQP

• TM

The image of YlpF

• The image was generated by PyMol

Sturucture Analysis

• 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

• 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

• 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 archeo-eukaryotic 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.