Based on the RbcX sequence conservation (Fig. 35 E and Fig. 36) several residues of Syn7002-RbcX were subjected to a mutational analysis in order to find out if they are of functional or structural importance. RbcX mutants were co-expressed with Syn7002- RbcL in E. coli and the resulting soluble lysates were analyzed for the presence of soluble RbcX and RbcL as well as for RbcL8 complex formation and potential activity (Fig. 40).
As described above, some mutations (L14E, L14E/V90E, R75L/E76A and ∆84-134) severely impaired dimer formation and caused RbcX to become insoluble or only poorly soluble (Fig. 40 B and C, triangles). Consequently, they could not support the production of soluble RbcL (Fig. 40 A, triangles), resulting in a situation resembling the expression of RbcL alone (Fig. 40 A, lane 1). All other RbcX mutants were expressed as soluble proteins and since their migration pattern in Native PAGE was similar to wildtype RbcX (except for the smaller deletion mutants), the above described dimer structure can be assumed to be valid for all of them (Fig. 40 B and C). Several RbcX mutations did not considerably affect the solubility or assembly of Syn7002-RbcL (Fig. 40 A, circles). However, the mutational analyses confirmed the importance of the conserved regions of RbcX as potential interaction sites between RbcX and RbcL: the central groove of the dimer and the peripheral polar regions around the corners of the molecule.
On the one hand, the double mutant Y17A/Y20L prevented the formation of soluble RbcL almost completely (Fig. 40 A, square). When only the single tyrosines were
RESULTS 109
mutated, solubility and assembly of RbcL were reduced, but not abolished (Fig. 40 A, lanes 6-8). Hence, the functional cooperation of both Y17 and Y20 is required for proper operation of RbcX. Y17 and Y20 form in concert with I50 a highly conserved hydrophobic area which is located on the surface of each protomer in the central groove of the RbcX dimer (Fig. 35 E). With a central access of 5.4 Å, this groove is wide enough to accommodate a polypeptide chain in an extended conformation and it thus represents a potential site for the interaction of RbcX with an unstructured peptide of RbcL.
On the other hand, the mutations Q29A, E32A, R70A and R102A allowed the accumulation of soluble RbcL but did not support its proper assembly to RbcL8 cores, as
indicated by Native PAGE and by the absence of RuBisCO activity upon supplementation with RbcS (Fig. 40 A, asterisks). All these critical residues are located in conserved polar surface regions around the edges of the RbcX dimer (Fig. 35 E). These areas are formed by helices α4 and by the turn region between helices α1 and α2. They might be suitable for interaction with the corresponding polar surface of the RbcL subunit.
Mutation of other residues which are located within or in close proximity to these two regions (e.g. T13, T33, N34, E76, E107 or ∆106-134) were without measurable effect, presumably due to minor or different functional relevance.
Since the crystal structure of three Syn7002-RbcX mutants (Y17A/Y20L, Q29A and R70A) was nearly identical to that of wildype RbcX (Table A6), the functional significance of both potential protein-protein interaction areas (central groove and peripheral corner surfaces) is assignable to both mutant and wildtype RbcX.
Figure 40. Functional analysis of Syn7002-RbcX mutants.
(A) Syn7002-RbcL was co-expressed in E. coli with wildtype or mutant Syn7002-RbcX as indicated. Soluble cell lysate was analyzed for soluble RbcL by 16 % SDS-PAGE and for RbcL8 complex assembly by 6 % Native PAGE, followed by immunoblotting against RbcL. Carboxylation activity in the soluble cell lysate was measured upon addition of purified Syn6301-RbcS. Syn7002-RbcL expressed without RbcX (1) or with wildtype RbcX (2) served as negative or positive control, respectively. (o) RbcX mutants similar to wt RbcX, supporting synthesis of soluble RbcL and properly assembled RbcL8 with carboxylation activity in presence of RbcS. (∗) RbcX mutants supporting production of soluble RbcL but not or hardly their assembly to RbcL8 complexes. (∆) RbcX mutants that are not or very poorly soluble themselves and therefore not or only poorly supporting production of soluble RbcL. (□) RbcX is soluble but not competent to allow production of soluble RbcL.
(B-C) Soluble cell lysate from expressions in (A) was analyzed by 17.5 % SDS-PAGE (B) and by 6 % Native PAGE (C), followed by Coomassie blue staining. Indicated RbcX mutants (∆) were insoluble or poorly soluble and thus mainly found in the pellet fraction (not shown).
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In order to characterize the interaction of Syn7002-RbcL with wildtype or mutant Syn7002-RbcX, RbcL was co-expressed in E. coli with N-terminally FLAG-tagged RbcX and soluble cell lysates were subjected to gel filtration chromatography.
When Syn7002-RbcL was co-expressed with functionally unimpaired wildtype Syn7002- RbcXFLAG, the majority of soluble RbcL fractionated as complex of approximately 400
kDa, consistent with the elution pattern of purified Syn6301-RbcL8 cores. Assembly
intermediates, such as RbcL monomers or dimers, could not be detected, suggesting that assembly of RbcL subunits into L8 cores is a very efficient process. Some RbcL eluted in
very early fractions and was most likely misassembled, aggregated RbcL (> 800 kDa), migrating with a molecular weight behond GroEL (Fig. 41 A, i). RbcX did not co- fractionate with RbcL by gel filtration, suggesting that the interaction between Syn7002- RbcL and Syn7002-RbcXFLAG must be dynamic. However, the interaction between
Syn7002-RbcL and Syn7002-RbcXFLAG was confirmed by co-immunoprecipitation from
the soluble cell lysates with ANTI-FLAG affinity beads. Precipitated proteins were eluted from the immunobeads under native conditions with excess of 3x FLAG-peptide and subsequently subjected to gel filtration. RbcL fractionated as RbcL8 complex with no
misassembled, aggregated or monomeric RbcL observed, indicating that wildtype RbcX interacts with Syn7002-RbcL8 cores (Fig. 41 B, i). However, despite their interaction
during the immunoprecipitation, RbcX again failed to co-fractionate with RbcL. This observation suggests that the interaction between RbcL and RbcX is rather labile and susceptible to disruption by gel filtration.
When Syn7002-RbcL was co-expressed with Syn7002-RbcX(Q29A)FLAG, only
misassembled or aggregated RbcL was observed migrating in high molecular weight fractions upon gel filtration of soluble cell lysate (Fig. 41 A, ii). This is in agreement with the observation that mutations in the peripheral polar surface areas of RbcX dimers do not support the proper assembly of soluble RbcL into RbcL8 complexes, as shown by
Native PAGE (Fig. 40 A, asterisks). No association between RbcL and Syn7002- RbcX(Q29A)FLAG could be detected by immunoprecipitation (Fig. 41 B, ii), confirming
that the assembly failure by Syn7002-RbcX(Q29A)FLAG is probably due to unstable
interaction between RbcL and RbcX, leading to misassembly and/or aggregation of RbcL.
Syn7002-RbcX(Y17A)FLAG, a mutant with a moderately impaired function of the central
groove, resulted (besides some aggregated RbcL) in a reduced formation of assembled RbcL8 and inefficient co-immunoprecipitation (Fig. 41 A and B, iii).
Based on the results depicted in Fig. 40 and 41, two distinct regions on RbcX are necessary for productive interaction with RbcL. The functional integrity of the central binding groove is a prerequisite for the production of soluble and thus assembly- competent RbcL. On the other hand, the polar peripheral areas at the edges of RbcX dimers are crucial for the proper assembly of soluble RbcL into RbcL8 cores.
Figure 41. Functional analysis of Syn7002-RbcX-mutants.
Syn7002-RbcL was co-expressed with Syn7002-RbcXFLAG (i), Syn7002-RbcX(Q29A)FLAG (ii) or Syn7002-RbcX(Y17A)FLAG (iii) in E. coli.
(A) Soluble cell lysates were subjected to gel filtration (Superdex 200, 10/30) in 50 mM Tris-HCl, pH 8, 50 mM NaCl, 5 mM MgCl2. Fractions were analyzed for RbcL and RbcXFLAG by SDS-PAGE, followed by immunoblotting. The elution pattern for GroEL (ca. 800 kDa) and Syn6301-RbcL8 (ca. 400 kDa) is indicated.
(B) Soluble cell lysates were subjected to co-immunoprecipitation of RbcL with anti-FLAG affinity beads. Pulled down proteins were eluted under native conditions with excess of 3x FLAG peptide and were analyzed by gel filtration as described in (A).
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