title: The proteolysis adaptor, NblA, binds to the N-terminus of β-phycocyanin: Implications for the mechanism of phycobilisome degradation.
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 BS3cross-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 K52in NblA is cross-linked to T2in 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-K44and M1-K52cross-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.
authors.affiliation: Department of Biology, Washington University, Campus Box 1095, One Brookings Drive, St. Louis, MO, 63130-4899, USA.|US Environmental Protection Agency, 1200 Pennsylvania Ave, NW (MC-7403M), Washington, DC, 20460, USA.
authors.forename: Amelia Y
authors.initials: AY
authors.lastname: Nguyen
authors.affiliation: Laboratory for Computational Biology & Biophysics, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
authors.forename: William P
authors.identifier: http://orcid.org/0000-0001-9724-0905
authors.initials: WP
authors.lastname: Bricker
authors.affiliation: Department of Chemistry, Washington University, St. Louis, MO, 63130, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Biology, Washington University, Campus Box 1095, One Brookings Drive, St. Louis, MO, 63130-4899, USA.|Department of Chemistry, Washington University, St. Louis, MO, 63130, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Weisz
authors.affiliation: Department of Chemistry, Washington University, St. Louis, MO, 63130, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Gross
authors.affiliation: Department of Biology, Washington University, Campus Box 1095, One Brookings Drive, St. Louis, MO, 63130-4899, USA. pakrasi@wustl.edu.
authors.forename: Himadri B
authors.identifier: http://orcid.org/0000-0001-8240-2123
authors.initials: HB
authors.lastname: Pakrasi
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D045524:Phycobilisomes / Q000378:metabolism; D010798:Phycocyanin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D046940:Synechococcus / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D045524:Phycobilisomes; D011993:Recombinant Fusion Proteins; D010798:Phycocyanin
keywords: Cross-linking; Cyanobacteria; Mass spectrometry; Nitrogen starvation; Phycobilisome proteolysis; Protein docking