Unlike its eukaryotic isoform, RbcL from Synechococcus sp. PCC6301 are compatible with E. coli GroEL chaperonins for proper folding, followed by assembly with RbcS into holoenzyme without additional chaperones. Here we show that sections 4 and 5 are not essential for GroEL interaction as substitution with corresponding Chlamydomonas sequences still allowed folding and assembly into their final hexadecameric complexes (L8S8) in E. coli (Fig. 4), although it is worth mentioning that Chlamydomonas sequences introduced into sections 4 and 5 chimeras might have changed their kinetic properties. Notably, section 4 chimera showed reduced amount of L8S8 than wild-type Rubisco and section 5 chimera. Section 4 chimera has two amino acid substitutions, S395A and V396C. One possibility is that these substitutions affect the expression level of RbcL and RbcS. But looking at the SDS-PAGE and Western blot analysis of total cellular extracts, only slight differences in the expressed level of RbcL and RbcS were observed among wild-type and chimeric Rubiscos (Fig. 3). This is consistent with previous findings whereby most mutations in RbcL generally do not affect the steady-state mRNA level [22, 34]. Another possibility is that S395A and/or V396C reduced the thermal stability of the holoenzyme, as have been reported before for other substitutions in RbcL [10, 39, 40].
As sections 1, 2, 3 and 6 chimeras showed no detectable L8S8 (Fig. 4), sections of Synechococcus RbcL that might be important for interacting with GroEL can be narrowed down to these sections, which comprise residues 248-272 (Section 1), 273-297 (Section 2), 348-372 (Section 3) and 423-447 (Section 6). Considering that GroEL chaperonin form multivalent interaction with its substrate polypeptide, whereby substrate polypeptides bind to multiple subunits of heptameric GroEL ring for efficient binding [41–43], it is possible that more than one of these sections interact with GroEL. Indeed, it has been reported that Rubiscos bind to a minimum of three consecutive subunits of GroEL ring for efficient binding [41, 43]. Binding of Rubisco to GroEL mutant with less than three consecutive binding-competent apical domain resulted in significantly reduced amounts of binary complexes of Rubisco and GroEL mutant, as compared to the wild-type GroEL [43]. In accordance with this finding, cryo-electron microscopy (cryo-EM) study of GroEL-RbcL binary complex showed the C-terminal domain of RbcL folding intermediate in contact with three consecutive apical domains while the N-terminal domain in contact with one apical domain [44]. Therefore, it may be possible that the three separate RbcL C-terminal regions (residues 248-297, 348-372 and 423-447) examined herein are involved in GroEL interaction and that loss of any favourable interactions with these three regions disrupted consecutive binding of RbcL to GroEL subunits. Consequently, binding between GroEL and RbcL was abolished or substantially reduced to an extent that only a small number of RbcLs were captured by GroEL, properly folded and assembled, resulting in the amount of L8S8 was undetectable.
In fact, by fitting the cryo-EM structure (EMD-6726) [44] and R. rubrum Rubisco (PDB ID 5RUB), it is found that residues 35-67, 89-116, 201-224, 236-253, 268-280, 422-457 of the R. rubrum RbcL might interact with GroEL. These residues correspond to Synechococcus RbcL residues 44-47, 104-125, 208-231, 243-260, 270-282, and 433-468. Only eleven of these residues (i.e. residues 252, 253, 259, 276, 279, 280, 435, 441, 442, 444 and 446) were changed among our unassembled section chimeras. Thus, these eleven residues could be of particular interest for mediating RbcL-GroEL binding.
Other than loss of GroEL interaction, non-assembly could be due to structural destabilizing effect of substitutions when changed to the Chlamydomonas counterpart. Therefore, despite of being captured by the GroEL chaperonin, the chimeric RbcL monomer might not fold into its native structure stably, and thus was unable to assemble into the final hexadecamer. Sections 1, 2, 3 and 6 chimeras have four to eight substitutions (Fig. 2). In general, amino acid substitutions could contribute to beneficial, neutral or deleterious effects [45]. In the case of our chimeras, some of these substitutions might have disrupted intra-subunit interactions or even exerted disturbances to the tertiary structure. For section 1 chimera, which has five substitutions, A269G has been reported to improve the fitness of Rubisco-dependent E. coli [22]. Therefore, non-assembly is more likely caused by the remaining four substitutions. As for section 6 chimera, one of the six mutations, E444S, could cause loss of intra-subunit salt-bridge with R432 in the RbcL monomer (Fig. 5a).
In addition, as there are three states of assembly: dimeric L2 pair, (L2)4 core complex and final hexadecameric L8S8, mutations that affect the inter-subunit interactions of any of these assembled complexes could eventually lead to non-assembly. Of all mutations involved in non-assembly, only E348D in section 3 chimera is located at the interface between RbcL and RbcS. By comparing the Rubisco crystal structures of Synechococcus (PDB ID 1RBL) and Chlamydomonas (PDB ID 1GK8), this mutation could result in replacement of the carboxyethyl side-chain of Glu-348 by a shorter carboxymethyl side-chain of Asp, which could potentially disrupt the salt-bridge with Lys-96 in the small subunit [20] (Fig. 5b).