Structural und functional characterization of Rubisco assembly chaperones

In the present study, the structure and mechanism of two assembly
chaperones of Rubisco, Raf1 and RbcX, were investigated. The role
of Raf1 in Rubisco assembly was elucidated by analyzing
cyanobacterial and plant Raf1 with a vast array of biochemical and
biophysical techniques. Raf1 is a dimeric protein. The subunits
have a two-domain structure. The crystal structures of two separate
domains of Arabidopsis thaliana (At) Raf1 were solved at
resolutions of 1.95 Å and 2.6–2.8 Å, respectively. The oligomeric
state of Raf1 proteins was investigated by size exclusion
chromatography connected to multi angle light scattering (SEC-MALS)
and native mass spectrometry (MS). Both cyanobacterial and plant
Raf1 are dimeric with an N-terminal domain that is connected via a
flexible linker to the C-terminal dimerization domain. Both Raf1
poteins were able to promote assembly of cyanobacterial Rubisco in
an in vitro reconstitution system. The homologous cyanobacterial
system resulted in very high yields of active Rubisco (>90%),
showing the great efficiency of Raf1 mediated Rubisco assembly. Two
distinct oligomeric complex assemblies in the assembly reaction
could be identified via native PAGE immunoblot analyses as well as
SEC-MALS and native MS. Furthermore, a structure-guided mutational
analysis of Raf1 conserved residues in both domains was performed
and residues crucial for Raf1 function were identified. A new model
of Raf1 mediated Rubisco-assembly could be proposed by analyzing
the Raf1-Rubisco oligomeric complex with negative stain electron
microscopy. The final model was validated by determining
Raf1-Rubisco interaction sites using chemical crosslinking in
combination with mass spectrometry. Taken together, Raf1 acts
downstream of chaperonin-assisted Rubisco large subunit (RbcL)
folding by stabilizing RbcL antiparallel dimers for assembly into
RbcL8 complexes with four Raf1 dimers bound. Raf1 displacement by
Rubisco small subunit (RbcS) results in holoenzyme formation. In
the second part of this thesis, the role of eukaryotic RbcX
proteins in Rubisco assembly was investigated. Eukaryots have two
distinct homologs of RbcX, RbcX-I and RbcX-II. Both, plant and
algal RbcX proteins were found to promote cyanobacterial Rubisco
assembly in an in vitro reconstitution system. Mutation of a
conserved residue important for Rubisco assembly in cyanobacterial
RbcX also abolished assembly by eukaryotic RbcX, underlining
functional similarities among RbcX proteins from different species.
The crystal structure of Chlamydomonas reinhardtii (Cr) RbcX was
solved at a resolution of 2.0 Å. RbcX forms an arc-shaped dimer
with a central hydrophobic cleft for binding the C-terminal
sequence of RbcL. Structural analysis of a fusion protein of CrRbcX
and the C-terminal peptide of RbcL suggests that the peptide
binding mode of CrRbcX may differ from that of cyanobacterial RbcX.
RbcX homologs appear to have adapted to their cognate Rubisco
clients as a result of co-evolution. Preliminary analysis of RbcX
in Chlamydomonas indicated that the protein functions as a Rubisco
assembly chaperone in vivo. Therefore, RbcX was silenced using RNAi
in Chlamydomonas which resulted in a photosynthetic growth defect
in several transformants when grown under light. RbcX mRNA levels
were highly decreased in these transformants which resulted in a
concomitant decrease of Rubisco large subunit levels. Biochemical
and structural analysis from both independent studies in this
thesis show that Raf1 and RbcX fulfill similar roles in Rubisco
assembly, thus suggesting that functionally redundant factors
ensure efficient Rubisco biogenesis.