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overall architecture seems to be conserved among cyanobacteria (Fig. 33, 36, 37 and 39). The dimer forms an arc-shaped complex with a central diagonal groove which could be shown to be capable of binding the C-terminus of RbcL subunits. This interaction requires the recognition motif EIKFEFDxxD in the RbcL C-terminus (residues 459 to 468 in Syn7002-RbcL). This peptide binds to RbcX in an extended conformation with the side chains F462 and F464 extending into conserved hydrophobic cavities of the groove, supported by hydrogen bonds and van-der-Waals contacts (Fig. 42 and 43).

Mutation of critical residues in the binding groove of RbcX resulted in disruption or attenuation of the RbcL-RbcX interaction and in misassembly or aggregation of RbcL (Fig. 40, 41 iii and 49 D). The same effect had C-terminal deletion or mutation of RbcL subunits, irrespective if they represented RbcX-dependent (Syn7002-RbcL) or RbcX- independent (Syn6301-RbcL) assembly types (Fig. 44, 45 and 49 F-H). This indicates on the one hand, that functional integrity of the RbcX binding groove is a prerequisite for the interaction with RbcL in order to keep it soluble and assembly-competent. On the other hand, integrity of the RbcL C-terminus with the recognition motif (particularly the presence of the two included phenylalanines) is not only critical for the initial recognition by and subsequent interaction with RbcX, but is itself required for RbcL assembly- competence and/or stabilization of assembly intermediates or RbcL8 cores.

The C-terminal RbcL peptide is located on the outer surface of assembled RuBisCO and contributes to the structural regulation of its catalysis. Binding of ligands (substrates or inhibitors) to the empty active site of RuBisCO triggers the closure of the active site and conformational changes in the C-terminal strand. The latter then stabilizes the closed position of the active site`s lid and is itself fastened in the closed state by a latch which involves D468 of the RbcX recognition motif (Duff et al. 2000). During RbcL assembly the C-terminal strand might be more detached from the RbcL subunits and the hydrophobic side chains might be more solvent exposed. Binding of RbcX could therefore protect and stabilize the C-terminus and thus the whole protein during assembly, thereby preventing uncontrolled misassembly or aggregation.

DISCUSSION 139

In addition to the central groove, a second region on RbcX with functional relevance for productive interaction with RbcL is formed by the polar surface areas around edges of the RbcX dimer. Mutation of critical conserved residues in these regions resulted in formation of soluble but misassembled RbcL complexes of high molecular weight (Fig. 40 and 41 ii). This implies that the peripheral corner regions of RbcX are essential to coordinate the proper positioning and suitable contact formation between the RbcL subunits of assembly intermediates, thereby allowing or enforcing the formation of RbcL8

cores which are convertible into active RbcL8S8 holoenzymes.

The combination of both, the central binding cleft and the polar peripheral regions of RbcX, appears to enable RbcX to accomplish the proper assembly of RbcL subunits. Anchoring of the RbcL C-terminus in the binding cleft is required to keep RbcL in a soluble state and to secure RbcL in an orientation relative to RbcX which is optimal for interaction with the peripheral RbcX binding surfaces. On this structural basis, optimal positioning and interconnection of the RbcL subunits is possible. In the resulting RbcL8

complex, RbcX might be bridging adjacent RbcL dimers and masking parts of the surface which is covered by the globular domain of RbcS in the final holoenzyme complex (Fig. 51). Such overlapping binding areas of RbcX and RbcS could explain why Syn7002- RbcX cannot re-bind to RbcL after its displacement by RbcS upon RbcL8S8 holoenzyme

assembly (Fig. 46 B, i and C, iii). Although RbcS in RbcL8S8 does not cover the C-

teminal RbcL peptide, it is also possible, that binding of RbcS induces conformational conversions in RbcL8. These could stabilize the RbcL C-terminus so as to make the

critical phenylalanine inaccessible to RbcX. The latter scenario could also prevent re- binding of RbcX either by itself or in concert with the steric overlap of recognition and binding regions for RbcS and RbcX.

Figure 51. Crystal structure of Syn6301-RbcL8S8 and model of the RbcL8(X2)8-complex. (A) RuBisCO of Synechococcus sp. PCC6301 bound to CABP (Newman et al. 1993). The RbcL subunits are shown in surface presentation, with the exception of the C-terminal peptides (amino acids 460-475), which are presented as blue coils. This conformation is found only in complex with the enzymatic substrate. Subunits of RbcL dimers are colored white and pale blue and residues interacting with RbcS (green ribbons) are depicted in red.

(B) Hypothetical model of RbcX binding to the assembled RbcL8 core complex using the same representation as in (A). The model is based on the structures of the Syn6301-RbcL8S8 complex and the RbcX dimer. One edge of RbcX fills the gap between adjacent RbcL dimers, masking most of the surface covered by the globular domain of RbcS in the holoenzyme complex. In this arrangement, the C-termini of RbcL (in red) kink horizontally immediately N-terminal to the recognition motif, leaving the secondary structure composition of RbcL intact. The figure was prepared by Dr. Andreas Bracher.