Crystal and solution studies of the "Plus-C" odorant-binding protein 48 from Anopheles gambiae: control of binding specificity through three-dimensional domain swapping.

Much physiological and behavioral evidence has been provided suggesting that insect odorant-binding proteins (OBPs) are indispensable for odorant recognition and thus are appealing targets for structure-based discovery and design of novel host-seeking disruptors. Despite the fact that more than 60 putative OBP-encoding genes have been identified in the malaria vector Anopheles gambiae, the crystal structures of only six of them are known. It is therefore clear that OBP structure determination constitutes the bottleneck for structure-based approaches to mosquito repellent/attractant discovery. Here, we describe the three-dimensional structure of an A. gambiae "Plus-C" group OBP (AgamOBP48), which exhibits the second highest expression levels in female antennae. This structure represents the first example of a three-dimensional domain-swapped dimer in dipteran species. A combined binding site is formed at the dimer interface by equal contribution of each monomer. Structural comparisons with the monomeric AgamOBP47 revealed that the major structural difference between the two Plus-C proteins localizes in their N- and C-terminal regions, and their concerted conformational change may account for monomer-swapped dimer conversion and furthermore the formation of novel binding pockets. Using a combination of gel filtration chromatography, differential scanning calorimetry, and analytical ultracentrifugation, we demonstrate the AgamOBP48 dimerization in solution. Eventually, molecular modeling calculations were used to predict the binding mode of the most potent synthetic ligand of AgamOBP48 known so far, discovered by ligand- and structure-based virtual screening. The structure-aided identification of multiple OBP binders represents a powerful tool to be employed in the effort to control transmission of the vector-borne diseases.

Rubisco Assembly in the Chloroplast.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step in the Calvin-Benson cycle, which transforms atmospheric carbon into a biologically useful carbon source. The slow catalytic rate of Rubisco and low substrate specificity necessitate the production of high levels of this enzyme. In order to engineer a more efficient plant Rubisco, we need to better understand its folding and assembly process. Form I Rubisco, found in green algae and vascular plants, is a hexadecamer composed of 8 large subunits (RbcL), encoded by the chloroplast genome and 8 small, nuclear-encoded subunits (RbcS). Unlike its cyanobacterial homolog, which can be reconstituted in vitro or in E. coli, assisted by bacterial chaperonins (GroEL-GroES) and the RbcX chaperone, biogenesis of functional chloroplast Rubisco requires Cpn60-Cpn20, the chloroplast homologs of GroEL-GroES, and additional auxiliary factors, including Rubisco accumulation factor 1 (Raf1), Rubisco accumulation factor 2 (Raf2) and Bundle sheath defective 2 (Bsd2). The discovery and characterization of these factors paved the way for Arabidopsis Rubisco assembly in E. coli. In the present review, we discuss the uniqueness of hetero-oligomeric chaperonin complex for RbcL folding, as well as the sequential or concurrent actions of the post-chaperonin chaperones in holoenzyme assembly. The exact stages at which each assembly factor functions are yet to be determined. Expression of Arabidopsis Rubisco in E. coli provided some insight regarding the potential roles for Raf1 and RbcX in facilitating RbcL oligomerization, for Bsd2 in stabilizing the oligomeric core prior to holoenzyme assembly, and for Raf2 in interacting with both RbcL and RbcS. In the long term, functional characterization of each known factor along with the potential discovery and characterization of additional factors will set the stage for designing more efficient plants, with a greater biomass, for use in biofuels and sustenance.

Reconstitution of Pure Chaperonin Hetero-Oligomer Preparations in Vitro by Temperature Modulation.

Chaperonins are large, essential, oligomers that facilitate protein folding in chloroplasts, mitochondria, and eubacteria. Plant chloroplast chaperonins are comprised of multiple homologous subunits that exhibit unique properties. We previously characterized homogeneous, reconstituted, chloroplast-chaperonin oligomers in vitro, each composed of one of three highly homologous beta subunits from A. thaliana. In the current work, we describe alpha-type subunits from the same species and investigate their interaction with ß subtypes. Neither alpha subunit was capable of forming higher-order oligomers on its own. When combined with ß subunits in the presence of Mg-ATP, only the α2 subunit was able to form stable functional hetero-oligomers, which were capable of refolding denatured protein with native chloroplast co-chaperonins. Since ß oligomers were able to oligomerize in the absence of α, we sought conditions under which αß hetero-oligomers could be produced without contamination of ß homo-oligomers. We found that ß2 subunits are unable to oligomerize at low temperatures and used this property to obtain homogenous preparations of functional α2ß2 hetero-oligomers. The results of this study highlight the importance of reaction conditions such as temperature and concentration for the reconstitution of chloroplast chaperonin oligomers in vitro.

The Cpn10(1) co-chaperonin of A. thaliana functions only as a hetero-oligomer with Cpn20.

The A. thaliana genome encodes five co-chaperonin homologs, three of which are destined to the chloroplast. Two of the proteins, Cpn10(2) and Cpn20, form functional homo-oligomers in vitro. In the current work, we present data on the structure and function of the third A. thaliana co-chaperonin, which exhibits unique properties. We found that purified recombinant Cpn10(1) forms inactive dimers in solution, in contrast to the active heptamers that are formed by canonical Cpn10s. Additionally, our data demonstrate that Cpn10(1) is capable of assembling into active hetero-oligomers together with Cpn20. This finding was reinforced by the formation of active co-chaperonin species upon mixing an inactive Cpn20 mutant with the inactive Cpn10(1). The present study constitutes the first report of a higher plant Cpn10 subunit that is able to function only upon formation of hetero-oligomers with other co-chaperonins.

The complexity of chloroplast chaperonins.

Type I chaperonins are large oligomeric protein ensembles that are involved in the folding and assembly of other proteins. Chloroplast chaperonins and co-chaperonins exist in multiple copies of two distinct isoforms that can combine to form a range of labile oligomeric structures. This complex system increases the potential number of chaperonin substrates and possibilities for regulation. The incorporation of unique subunits into the oligomer can modify substrate specificity. Some subunits are upregulated in response to heat shock and some show organ-specific expression, whereas others possess additional functions that are unrelated to their role in protein folding. Accumulating evidence suggests that specific subunits have distinct roles in biogenesis of ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco).

P. falciparum cpn20 is a bona fide co-chaperonin that can replace GroES in E. coli.

Human malaria is among the most ubiquitous and destructive tropical, parasitic diseases in the world today. The causative agent, Plasmodium falciparum, contains an unusual, essential organelle known as the apicoplast. Inhibition of this degenerate chloroplast results in second generation death of the parasite and is the mechanism by which antibiotics function in treating malaria. In order to better understand the biochemistry of this organelle, we have cloned a putative, 20 kDa, co-chaperonin protein, Pf-cpn20, which localizes to the apicoplast. Although this protein is homologous to the cpn20 that is found in plant chloroplasts, its ability to function as a co-chaperonin was questioned in the past. In the present study, we carried out a structural analysis of Pf-cpn20 using circular dichroism and analytical ultracentrifugation and then used two different approaches to investigate the ability of this protein to function as a co-chaperonin. In the first approach, we purified recombinant Pf-cpn20 and tested its ability to act as a co-chaperonin for GroEL in vitro, while in the second, we examined the ability of Pf-cpn20 to complement an E. coli depletion of the essential bacterial co-chaperonin GroES. Our results demonstrate that Pf-cpn20 is fully functional as a co-chaperonin in vitro. Moreover, the parasitic co-chaperonin is able to replace GroES in E. coli at both normal and heat-shock temperatures. Thus, Pf-cpn20 functions as a co-chaperonin in chaperonin-mediated protein folding. The ability of the malarial protein to function in E. coli suggests that this simple system can be used as a tool for further analyses of Pf-cpn20 and perhaps other chaperone proteins from P. falciparum.