Abstract
Phycobilisome (PBS) complexes are massive light-harvesting apparati in cyanobacteria that capture and funnel light energy to the photosystem. PBS complexes are dynamically degraded during nutrient deprivation, which causes severe chlorosis, and resynthesized during nutrient repletion. PBS degradation occurs rapidly after nutrient step down, and is specifically triggered by non-bleaching protein A (NblA), a small proteolysis adaptor that facilitates interactions between a Clp chaperone and phycobiliproteins. Little is known about the mode of action of NblA during PBS degradation. In this study, we used chemical cross-linking coupled with LC-MS/MS to investigate the interactions between NblA and phycobiliproteins. An isotopically coded BS3 cross-linker captured a protein interaction between NblA and β-phycocyanin (PC). LC-MS/MS analysis identified the amino acid residues participating in the binding reaction, and demonstrated that K52 in NblA is cross-linked to T2 in β-PC. These results were modeled onto the existing crystal structures of NblA and PC by protein docking simulations. Our data indicate that the C-terminus of NblA fits in an open groove of β-PC, a region located inside the central hollow cavity of a PC rod. NblA may mediate PBS degradation by disrupting the structural integrity of the PC rod from within the rod. In addition, M1-K44 and M1-K52 cross-links between the N-terminus of NblA and the C-terminus of NblA are consistent with the NblA crystal structure, confirming that the purified NblA is structurally and biologically relevant. These findings provide direct evidence that NblA physically interacts with β-PC.
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Acknowledgements
We thank Drs. Aparna Nagarajan, Michelle Liberton, Haijun Liu, Mark Bathe, and all the members of the Pakrasi and Bathe laboratories for collegial discussions and advice. Funding for this study was provided by the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under award number DE-SC 0001035. AYN has been supported by an NSF Graduate Research Fellowship Grant Number DGE-1143954. Funding for WPB has been provided by Army Research Office MURI Award W911NF1210420. WPB also acknowledges the Office of Naval Research DURIP Awards N00014-13-1-0664 and N00014-15-1-2830, which funded the high-performance computational cluster in the Bathe laboratory used for protein modeling. Mass spectrometry was supported in part by the NIH, NIGMS Grant No. P41GM103422 to MLG. We also thank Drs. Antje Baier, Anne Karradt, and Wolfgang Lockau for the generous gifts of NblA plasmids.
Author contributions
AYN and HBP conceived the project; AYN, and HZ performed the experiments; AYN, DAW, and HZ analyzed the mass spectrometry data; WPB designed and implemented the docking models, and analyzed the docking modeling data; MLG and HBP contributed to the development of the measurement capabilities used; AYN, WPB, DAW, MLG, and HBP wrote the manuscript.
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Nguyen, A.Y., Bricker, W.P., Zhang, H. et al. The proteolysis adaptor, NblA, binds to the N-terminus of β-phycocyanin: Implications for the mechanism of phycobilisome degradation. Photosynth Res 132, 95–106 (2017). https://doi.org/10.1007/s11120-016-0334-y
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DOI: https://doi.org/10.1007/s11120-016-0334-y