Rubisco folding and oligomeric assembly: Detailed analysis of an assembly intermediate

To become biologically active, a protein must fold into a distinct
three-dimensional structure. Many non-native proteins require
molecular chaperones to support folding and assembly. These
molecular chaperones are important for de novo protein folding as
well as refolding of denatured proteins under stress conditions. A
certain subset of chaperones, the chaperonins, are required for the
folding of the enzyme ribulose-1,5-bisphosphate
carboxylase/oxygenase (Rubisco); furthermore, correct folding of
Rubisco is also aided by the Hsp70 chaperone system. Rubisco
catalyzes the initial step of CO2 assimilation in the
Calvin-Benson-Bassham (CBB) cycle. Unfortunately, this enzyme is
extremely inefficient, not only does it exhibit a slow catalytic
rate (three CO2 molecules fixed per second per Rubisco) but it also
discriminates poorly between the assimilation of CO2 and O2 to its
sugar-phosphate substrate ribulose-1,5-bisphosphate (RuBP), the
latter resulting in loss of photosynthetic efficiency. Due to these
inefficiencies, carbon fixation by Rubisco is the rate limiting
step of the CBB cycle. Photosynthetic organisms must produce
tremendous amounts of Rubisco to alleviate these shortcomings;
therefore significant quantities of nitrogen stores are invested in
the production of Rubisco making Rubisco the most abundant protein
on earth. These drawbacks of Rubisco have important implications in
increasing CO2 concentrations and temperatures in the context of
global warming. The ability to engineer a more efficient Rubisco
could potentially reduce photosynthetic water usage, increase plant
growth yield, and reduce nitrogen usage is plants. However,
eukaryotic Rubisco cannot fold and assemble outside of the
chloroplast, hindering advancements in creating a more efficient
Rubisco. Form I Rubisco, found in higher plants, algae, and
cyanobacteria, is a hexadecameric complex consisting of a core of
eight ~50 kDa large subunits (RbcL), which is capped by four ~15
kDa small subunits (RbcS) on each end. The discovery of a
Rubisco-specific assembly chaperone, RbcX, has lead to a better
understanding of the components necessary for the form I Rubisco
assembly process. RbcX is a homodimer of ~15 kDa subunits
consisting of four α- helices aligned in an anti-parallel fashion
along the α4 helix. RbcX2 functions as a stabilizer of folded RbcL
by recognizing a highly conserved C-terminal sequence of RbcL:
EIKFEFD, termed the C-terminal recognition motif. As has been
demonstrated by studies of cyanobacterial Rubisco, de novo
synthesized RbcL is folded by the chaperonins, whereupon RbcX2
stabilizes the folded RbcL monomer upon release from the folding
cavity and then assists in the formation of the RbcL8 core. RbcX2
forms a dynamic complex with RbcL8 and as a result, RbcX2 is
readily displaced by RbcS docking in an ATP-independent manner,
thereby creating the functional holoenzyme. However, the exact
mechanism by which RbcS binding displaces RbcX2 from the RbcL8 core
is still unknown. Furthermore, though much advancement has been
made in the understanding of form I Rubisco folding and assembly,
an exact and detailed mechanism of form I Rubisco assembly is still
lacking. The highly dynamic complex of RbcL/RbcX is critical for
the formation of the holoenzyme; however it has hindered attempts
to characterize critical regions of RbcL that interact with the
peripheral regions of RbcX2. An important observation arose when
heterologous RbcL and RbcX2 components interacted; a stable complex
could form enabling in depth characterization of the RbcL/RbcX2
interaction. In the present study, the detailed structural
mechanism of RbcX2-mediated cyanobacterial form I Rubisco assembly
is elucidated. To obtain molecular insight into the RbcX2-mediated
assembly process of cyanobacterial form I Rubisco, cryo-EM and
crystallographic studies in concert with mutational analysis were
employed by taking advantage of the high affinity interaction
between RbcL and RbcX2 in the heterologous system (Synechococcus
sp. PCC6301 RbcL and Anabaena sp. CA RbcX2). Structure guided
mutational analysis based on the 3.2 Å crystal structure of the
RbcL8/(RbcX2)8 assembly intermediate were utilized to determine the
precise interaction site between the body of RbcL and the
peripheral region of RbcX2. From these studies a critical salt
bridge could be identified that functions as a guidepoint for
correct dimer formation, and it was observed that RbcX2 exclusively
mediates Rubisco dimer assembly. Furthermore, the mechanism of
RbcX2 displacement from the RbcL8 core by RbcS binding was
elucidated as well as indications of how RbcS docking on the RbcL8
core is imperative for full form I Rubisco catalytic function by
stabilizing the enzymatically competent conformation of an
N-terminal loop of Rubisco termed the ‘60ies loop’. Finally,
initial attempts in in vitro reconstitution of eukaryotic Rubisco
are reported along with the characterization of Arabidopsis
thaliana RbcX2 binding to the C-terminal recognition motif of the
Rubisco large subunit from various species.