Bioinformatic Analysis of Ethylene response Protein 1 of Arabidopsis thaliana Columbia

1
Bioinformatic Analysis of Ethylene response
Protein 1 of Arabidopsis thaliana Columbia

• Group 5
• Speakers Abdul and William

2
Identification of the gene

• Blastn (EBI) search of a 240 bp fragment revealed
  100 identity to mRNA for the putative Pro-rich
  protein ETR-1, a putative ethylene receptor
• Gene located on chromosome 1

3
AA sequence analysis

• 738 residues, 78 kDa
• Amino acid sdequence (AtEnsembl)
• MEVCNCIEPQWPADELLMKYQYISDFFIAIAYFSIPLELIYFVKKSA
  VFPYRWVLVQFGAFIVLCGATHLINLWTFTTHSRTVALVMTTAKVLTAVV
  SCATALMLVHIIPDLLSVKTRELFLKNKAAELDREMGLIRTQEETGRHVR
  MLTHEIRSTLDRHTILKTTLVELGRTLALEECALWMPTRTGLELQLSYTL
  RHQHPVEYTVPIQLPVINQVFGTSRAVKISPNSPVARLRPVSGKYMLGEV
  VAVRVPLLHLSNFQINDWPELSTKRYALMVLMLPSDSARQWHVHELELVE
  VVADQVAVALSHAAILEESMRARDLLMEQNVALDLARREAETAIRARNDF
  LAVMNHEMRTPMHIIALSSLLQETELTPEQRLMVETILKSSNLLATLMND
  VLDLSRLEDGSLQLELGTFNLHTLFREVLNLIKPIAVVKKLPITLNLAPD
  LPEFVVGDEKRLMQIILNIVGNAVKFSKQGSISVTALVTKSDTRAADFFV
  VPTGSHFYLRVKVKDSGAGINPQDIPKIFTKFAQTQSLATRSSGGSGLGL
  AISKRFVNLMEGNIWIESDGLGKGCTAIFDVKLGISERSNESKQSGIPKV
  PAIPRHSNFTGLKVLVMDENGVSRMVTKGLLVHLGCEVTTVSSNEECLRV
  VSHEHKVVFMDVCMPGVENYQIALRIHEKFTKQRHQRPLLVALSGNTDKS
  TKEKCMSFGLDGVLLKPVSLDNIRDVLSDLLEPRVLYEGM
• Secondary structure prediction
  (nnPredict)
• -------------HHHHHHHHE---HHEEEE------HHH
  HHH--------EEEEEE---EEEEH--HHHEEHEEE-----HEEHHHHHH
  HHEEEHHHHHHHHHEHE-------H-HHHHHHH-H-HHHHHHH--E--HH
  H--HHHHHHHHHH--------H---HHHHHHHHHHHHHHH---------E
  EEHHHH-----------------EEEE------EE---------------
  --EEEEEEEEH-HHH----------------HHHHHHEE-------HHHH
  HHHHHHHHHHHHHHHHHHHHHHHHHHHHHHH-HHHHHHHHHHHHHHHHHH
  HHH--HHHHHH------HHHEHHHH---H-------HHHHHHHHHHHHHH
  HHHHHHHHHH---HH----HEEH--HHHHHHHHHHHHHH--HHH-----
  HEH--------E----HHHHHHHEEEHH-HHHHH-----EEEEEEE----
  -HH--EEE------EEEEEE--------------HHHHHHHHHHH-EEE-
  -----EEEEHHHHHHHHH---EEEE--------EEEHE-EE---------
  --------------------EEEEH-H----HHHHH-HEEE---EEEE--
  ---HHHHHHH--HHE--E---------HHHHHHHHHH---------HEEE
  ------------H------------------HHHH-------HHH---
  
• (H helix, E beta sheet)
• Annotated information (Gene AT1G66340, AtEnsembl)
• two-component response regulator
  Similar to prokaryote sensory transduction
  proteins.
• Contains a histidine kinase and a
  response regulator domain.
• Homodimer.
• Membrane component.

4
Functional domains of ETR-1

• N-terminal hydrophobic domain for ethylene
  binding and membrane attachment.
• C-terminal domain has homology to amylase
  inhibitors, lipid transfer and seed storage
  proteins. A structure of this domain shows it to
  be a 4-helical bundle in a right-handed
  superhelix with an internal cavity (SMART
  database)
• Putative N-myristoylation element (residues
  107-112) and Amidation site (residues 123-126)
• 3 predicted TM regions in N-terminus from
  hydropathy plot (TMPred)

5
Multiple alignment of related proteins

• Used the ETR1 sequence as a query for a second
  BLAST search which yielded a large number of hits
  with significant homology.
• The sequences of homologous proteins from rose,
  grape and brassica were aligned with the sequence
  of ETR-1 from Aradopsis thaliana using Clustal W
• Arabidopsis MEVCNCIEPQWPADELLMKYQYISDFFIAIAYF
  SIPLELIYFVKKSAVFPYRWVLVQFGA 60
• Brassica MEVCNCIEPQWPADELLMKYQYISDFFIAVA
  YFSIPLELIYFVKKSAVFPYRWVLVQFGA 60
• Rose ----------------------------------
  -------------------- -----------------------------
  -------------------------LVQFGA 6
• Grape MESCDCIDPPWPGDELLMKYQYISDFFIALA
  YFSIPLELIYFVKKSAVFPYRWVLVQFGA 6
• 
  
• Arabidopsis FIVLCGATHLINLWTFTTHSRTVALVMTTAKVL
  TAVVSCATALMLVHIIPDLLSVKTREL 120
• Brassica FIVLCGATHLINLWTFTTHSRTVALVMTTAKV
  LTAVVSCATALMLVHIIPDLLSVKTREL 120
• Rose FIVLCGATHLINLWTFSVHSRTVAIVMATAK
  VFTAAVSCATALMLVHIIPDLLSVKTREL 66
• Grape FIVLCGATHLINLWTFGMHSRNVEVVMTTAK
  VLTAVVSCVTALMLVHIIPDLLSVKTREL 120
• Arabidopsis FLKNKAAELDREMGLIRTQEETGRHVRMLTHEI
  RSTLDRHTILKTTLVELGRTLALEECA 180
• Brassica FLKNKAAELDREMGLIRTQEETGRHVRMLTHE
  IRSTLDRHTILKTTLVELGRTLALEECA 180
• Rose FLKNKAAELDREMGLIRTQEETGRHVRMLTH
  EIRSTLDRHTILKTTLVELGRILTLEECA 126
• Grape FLKNKAAELDREMGLIRTQEETGRHVRMLTHE
  IRSTLDRHTILKTTLIELGRTLSLEECA 180
• Arabidopsis LWMPTRTGLELQLSYTLRHQHPVEYTVPIQLPV
  INQVFGTSRAVKISPNSPVARLRPVSG 240

6
Structure prediction using Homology Modelling

• Using CPHModels 2.0 our query was matched to a
  hit with e value 10-5. The sequence of ETR-1 was
  found to be 39.3 identical to a soya bean
  protein whose structure is in the protein data
  base (1.8 Angstrom resolution) Hyp1
• A member of a family of plant lipid-transfer and
  hydrophobic proteins.
• Predicts a 4-helical bundle, but the model cannot
  be displayed as there is not the appropriate
  software.

7
Summary

• Annotation of C-domain function suggests
  putative role in transfer of lipids across
  membranes and into seeds and in mobilisation of
  sugars from glycogen during germination.
  supported by the functions of orthologous
  proteins and by sequence alignments
• Structure of C-terminal domain available
• hydropathy predictions, etc suggest that the
  N-terminal domain may be responsible for membrane
  attachment and binding to ethylene

• BUT BIOINFORMATICS SIMPLY PROVIDES AN HYPOTHESIS
  WHICH NEEDS TO BE VERIFIED BY EXPERIMENT!