Cyanobacteria play an important role in global carbon fixation – with the numerically dominant Synechococcus and Prochlorococcus contributing a significant fraction of total primary production.
At it’s core, Cyanobacteria have evolved a CO2 concentrating mechanism (CCM) to enhance the CO2 fixation activity of the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), thereby improving photosynthetic performance. The central component of the CCM is a self-assembling proteinaceous organelle, the carboxysome. Two types of carboxysome, α and β differ in gene organization and associated proteins. In contrast to the β- carboxysome little is known about the assembly process of the α-carboxysome. In particular, an absolutely conserved α-carboxysome protein, CsoS2, is of unknown function. Fei Cai et. al. from the University of California, Berkeley focus in this study on the CsoS2 protein in three model organisms and show that CsoS2 is vital for α-carboxysome biogenesis. The primary structure of CsoS2 appears tripartite, composed of an N-terminal, middle (M)-, and C-terminal region. Repetitive motifs can be identified in the N- and M-regions. Multiple lines of evidence suggest CsoS2 is highly flexible, possibly an intrinsically disordered protein.
3d structural studies were hindered by the lack of protein crystals. Instead, peptide arrays were used to study CsoS2 interactions with potential binding partners.
A library of overlapping peptides each shifted by one amino acid to span the entire sequence of MIT9313 CsoS2 was synthesized on the chip. Three identical libraries were each designated for one specific carboxysome protein in a given binding assay: RuBisCO holoenzyme, CsoS1 and CsoS1D.
Positive hits for RuBisCO holoenzyme are observed in all three regions of CsoS2; the binding patterns of CsoS1 and CsoS1D are similar with the exception of the M-region. A region spanning 23 amino acids (ranging from I673 to A695), located in the relatively conserved C-terminal region of CsoS2, is evidently a hot-spot for protein-protein interactions between CsoS2 and other carboxysome proteins.
Figure 1 – A raw image of the peptide array. After binding assay (CsoS1D and RuBisCO), scanning of the chip was performed at 635 nm for signal of Alexa 647. Artificial color was applied to facilitate imaging of signal intensities with blue for no binding to red for maximal binding.
Figure 2 – A Hidden Markov model (HMM) logo for all α-Cyanobacterial CsoS2 orthologs. For demonstration purposes only, a simplified presentation of results from the protein-binding assay against MIT9313 CsoS2 peptide array are mapped onto the logo. The starting position of peptides among all positive hits is marked with RuBisCO, CsoS1 or CsoS1D symbols only if the averaged signal intensity (1) ranks in the top 10 out of all positive hits or (2) is a local maximum with >5 sequential positive hits. The saturation of each symbol is relative to its fraction ratio to the maximum signal intensity (as 100% saturation) of all positive hits from a given binding assay.
The results indicate a large interaction site/pocket for RuBisCO and suggest that CsoS1 and CsoS1D interact with CsoS2 at this position within a much narrower site/pocket. Based on the authors’ results from bioinformatic, biophysical, genetic and biochemical approaches, including the peptide array scanning for protein-protein interactions, they propose a model for CsoS2 function and its spatial location in the α-carboxysome.
- Cai F, Dou Z, Bernstein SL, Leverenz R, Williams EB, Heinhorst S, Shively J, Cannon GC, Kerfeld CA. (2015) Advances in Understanding Carboxysome Assembly in Prochlorococcus and Synechococcus Implicate CsoS2 as a Critical Component. Life (Basel) 5(2):1141-71. [article]