Download Arabidopsis thaliana proteins involved in signalling pathways

Arabidopsis thaliana proteins involved in signalling pathways: structural and
functional studies
Marta Grzechowiak
Summary
Structural studies of WRKY transcription factors
The WRKY proteins are a large superfamily of transcription regulators of plant genes induced
upon pathogen infection and during certain stages of plant development. Their hallmark is strong
conservation of the DNA binding domain which contains an invariant WRKYGQK sequence and
zinc binding motif. However, the overall sequences of individual representatives are highly
divergent. So far there were only structural studies of DNA binding domain available.
The main goals of this thesis were structural studies of entire copies of the WRKY transcription
factors from Arabidipsis thaliana. In presented studies, I developed an efficient method for
expression and purification of recombinant AtWRKY50 and AtWRKY18DBD protein. The
obtained proteins retaining the biological activity of the DNA binding. The methods presented in
this study allow the production of a significant amount of AtWRKY50 and AtWRKY18 DBD in
bacterial expression system for further functional and structural studies. Obtained recombinant
proteins were high quality to carry out crystallization experiments however all attempts to obtain
well diffracting crystals of AtWRKY18DBD or AtWRKY50 protein failed, thus solwing highresilution crystallographic structure was impossible.
The CD spectrum and bioinformatics sequence analyses employed in this studies allowed to
deduce that AtWRKY50 lack of well defined secondary structure and is partially disordered.
This may explain difficulties in crystallization and failure to gain the main goal of the thesis solving the crystallographic structure of the protein of interests.
ITC and EMSA analyses provided evidence for activity of recombinant AtWRKY50 protein and
AtWRKY18DBD toward DNA binding.
Structural studies of enzymes involved in phosphate metabolism
Inorganic pyrophosphatase (PPase) is a ubiquitous cytosolic enzyme which catalyzes the
hydrolysis of inorganic pyrophosphate (PPi) to orthophosphate (Pi).
Arabidopsis thaliana inorganic pyrophosphatase (AtPPA1) coding DNA (ppa1 gene) was cloned
into bacterial expression vector and overproduced in E. coli cells as a fusion (His-tagged)
protein. The recombinant protein was purified from the bacterial lysate by two consecutive
chromatographic steps: chelating chromatography on Ni2+-charged resin followed by FPLC size
exclusion chromatography. The homogenous protein was submitted for crystallization. X-Ray
diffraction data extending to 1.9Å resolution were collected using synchrotron radiation. The
structure was solved by molecular replacement using Pyrococcus furiosus structure coordinates
(PDB code: 1twl) having the highest sequence identity to AtPP1 (49%) and refined to R-factor
below 15.6%. The structure coordinates of AtPPA1 have been deposited in PDB with code: 4lug.
The structure of AtPP1 represents an OB-fold which overlaps with other structural models for
known bacterial and yeast inorganic pyrophosphatases. PPases are oligomeric enzymes that are
active as homohexamers, or homotetramers composed of about 20 kDa subunits in prokaryotes.
Eukaryotic PPases act as homodimers with 30-35 kDa subunits. Plant PPase is an exception
because it function as 75 kDa trimer. Moreover, the analysis of AtPPA1 sequence using PsiPred
(signal peptide predictor) revealed that it posses N-terminal putative transit peptide of
mitochondrial targeting, and a possible cleavage site at Val31. In vitro, cleavage of short (few
kDa) fragment is observed during protein storage. Mutant with substitution D98N shows delayed
autoproteolysis compared to wild type (WT) protein. Crystal structure refinement and protein
sequencing revealed that the N-terminal fragment corresponding to the predicted mitochondrial
targeting peptide is cleaved.