TÍTULO DE LA PROPUESTA.

The assembly of some multi-domain/oligomeric protein complexes requires the assistance of particular assembly chaperones. The necessity of assembly chaperones for support in the formation of the nucleosome (Laskey et al. 1978) and the proteasome (Witt et al. 2000) has been well established. The term molecular chaperone was initially coined to define the set of proteins involved in assisting the assembly of oligomeric complexes, and the term was later broadened to include proteins involved in the folding of non-native proteins

(Ellis 2006). As described in section 2.2, there are many examples of chaperones essential for the folding of non-native proteins but there are few examples of assembly chaperones apart from their role in nucleosome and proteasome formation and in the assembly of form I Rubisco.

Recently, the discovery of a new ATP-independent chaperone, Spy, located in the periplasm of E. coli was reported (Quan et al. 2011). The authors have demonstrated that this cradle shaped protein assists with folding of unstable proteins and suppresses protein aggregation. There is a striking structural resemblance between the Spy protein and RbcX2

(Figure 5.1). The Spy protein is a homodimer of subunits with four α-helices, aligned in an anti-parallel fashion along the α3 helix, and the dimer is about 30 kDa in size. It has a concave ‘groove-like’ region that has hydrophobic patches. The Spy protein was shown to be induced by protein conformational state, and could also act as a chaperone for a variety of substrates

in vitro. However, the exact mechanism of its function remains to be determined especially since this protein acts ATP-independently and it is unknown how it is released from substrate after binding. RbcX2 also acts in an ATP-independent manner; however it is known to be

released from its substrate by the binding of the small subunit to the RbcL8/(RbcX2)8 complex

in a mechanism involving the structuring of the RbcL ‘60ies loop’ (section 4.3.2.3 and 5.2.1). RbcX2 also operates as an aggregation suppressor, and the folding intermediates of Rubisco

are highly aggregation prone (Gutteridge and Gatenby 1995). However, there has been no known report of RbcX2 induction by unfolded protein or stress conditions; on the contrary,

RbcX2 is of low abundance and difficult to detect in cellular lysate (Emlyn-Jones et al. 2006).

Nevertheless, a recent proteomics study was able to detect trace amounts peptides of both RbcX2 isoforms in the stroma of A. thaliana (Olinares et al. 2010), indicating that RbcX2

protein is expressed in plants and transported to the chloroplast, albeit at low levels.

Since RbcX2 is not found at very high concentrations, its function may be more

important during plant development when high amounts of Rubisco are being synthesized. Furthermore, Rubisco is a highly stable, long-lived protein in its holoenzyme form, making it unnecessary to constantly have RbcX2 protein present once Rubisco is made. Nevertheless,

the newly described periplasmic E. coli chaperone, Spy, could provide clues as to other possible functions of eukaryotic RbcX2. An important step to understanding the function of

eukaryotic RbcX2 would be the analysis of the crystal structure. Unfortunately, this protein is

highly aggregation prone above concentrations of 1 mg/ml (section 3.4.3.6), making crystallization difficult.

Figure 5.1 Comparison of the Spy protein crystal structure with RbcX2 crystal structure.

(A) Front and side ribbon representation of the Spy protein crystal structure with subunits indicated in light blue and magenta. The α-helices 1-4 are labeled along with the N- and C-termini. Adapted from: (Quan et al. 2011). (B) Front and side

ribbon representation of Syn7002-RbcX2 crystal structure with subunits indicated in light brown and yellow. The N- and C-

termini are labeled.

5.3.1

Mechanism of RbcX

-mediated Rubisco assembly

In this study the detailed structural mechanism of RbcX2-mediated cyanobacterial

form I Rubisco assembly has been elucidated (section 4.3.2.3). The chaperone-assisted folding and assembly of form I Rubisco are tightly coupled; RbcX2 binds RbcL immediately

after encapsulation and release from GroEL/ES to ensure the formation of assembly- competent intermediates. RbcX2 recognizes and binds the C-terminal of RbcL (area I) along

with area II (Figure 4.15). Binding of RbcX2 to RbcL prevents rebinding to chaperonin and

supports RbcL dimerization, possibly by ordering of the C-terminal helices of RbcL (Figure 4.8). Furthermore, RbcX2 promotes RbcL dimerization by salt bridge formation to area III of

the adjacent RbcL subunit.

RbcL dimerizes in an anti-parallel fashion by alignment of three charge pairs. Two charge pairs are located at the RbcL/RbcL interface, while the additional charge pair is contributed by the bound RbcX2 (Figure 4.16 and Figure 4.17). These guide-points are

necessary to prevent misalignment and off-pathway aggregation by formation of the intermediate RbcL2/(RbcX2)2. This intermediate is less prone to aggregation due to the burial

of the hydrophobic surfaces located at the RbcL/RbcL interface (Figure 4.19 and Figure 4.20). The intermediate can then assemble to RbcL8/(RbcX2)8 by surface shape complementarity and

polar contacts at the RbcL2/RbcL2 interface. RbcX2 bound to the RbcL8 core promotes

formation of the RbcS binding interface by ordering the C-terminal helices of RbcL (Figure 4.8). RbcS then binds in between RbcL dimers of the RbcL8 core, which leads to structuring

of the RbcL ‘60ies loop’. The structuring of the ‘60ies loop’ in turn blocks RbcX2 binding to

area III of RbcL and mediates RbcX2 displacement. When the ‘60ies loop’ is structured, RbcL

is rendered sterically incompatible with RbcX2 binding. Figure 5.2 provides a structural

description of the mechanism of RbcX2-medaited Rubisco assembly.

A

B

C

C N N

Figure 5.2 Model of RbcX2-assisted Rubisco assembly.

RbcL is encapsulated and folded by GroEL/ES. (1) RbcX2 binds the C-terminal recognition motif of RbcL (area I) and area II

upon release from chaperonin. (2) RbcL dimerization. The indicated complementary surface charge guide-points from RbcL

and RbcX2 most likely play a role in ensuring proper alignment of the anti-parallel dimer. RbcX2 forms a salt bridge with

area III of the adjacent RbcL subunit and stabilizes the dimers by acting as a ‘molecular staple’. (3) The intermediates,

RbcL2/(RbcX2)2, assemble to the RbcL8/(RbcX2)8 complex, where RbcX2 also likely supports formation of the RbcS binding

interface on RbcL. (4) Binding of RbcS causes the structuring of the RbcL ‘60ies loop’ causing steric blockage of RbcX2

binding to area III on RbcL. This leads to the displacement of RbcX2 and formation of the functional Rubisco holoenzyme.

Figure prepared by Dr. Andreas Bracher.