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Analysis of pyrenoid proteome in the diatom Phaeodactylum tricornutum

ABSTRACT: Diatoms are a major primary producer accounting for 20% of the global primary production. Their massive carbon fixation is driven by the CO2-concentrating mechanisms (CCM), in which CO2 is actively supplied to the Rubisco localized in the pyrenoid, a membrane-less organelle of the plastid. To promote CO2 fixation, CO2 concentration in the pyrenoid matrix is assumed to be elevated while leakage of CO2 is minimized. However, due to the lack of information on the structural basis of diatom pyrenoid, the mechanism allowing efficient CO2 supply in conjunction with pyrenoid structure remains to be elucidated. While isolation of diatom pyrenoid is technically difficult due to structural instability, here we applied in vivo crosslinking using photo amino acids (pAA) which enabled us to obtain Rubisco enriched fractions. Based on LC-MS/MS analysis of the RbcL-enriched fraction, we identified a novel protein that surrounds the pyrenoid like a shell and hence named a Pyshell (Pyrenoid shell). Using genome-edited mutants, Pyshell was demonstrated to be a critical component required for growth under CO2-limiting conditions. The comprehensive analysis and identification of the pyrenoid component gave an important insight into the molecular basis driving the oceanic primary production.

ORGANISM(S): Phaeodactylum Tricornutum

SUBMITTER: Yusuke Matsuda

PROVIDER: PXD041920 | JPOST Repository | Sat May 04 00:00:00 BST 2024

REPOSITORIES: jPOST

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Project description:PRIDE > Archive > PXD013420 Project PXD013420 Download Project Files Summary Title A binding site hotspot map of the FKBP12–rapamycin–FRB ternary complex by photo-affinity labeling and mass spectrometry-based proteomics Description Structural characterization of small molecule binding site hotspots within the global proteome is uniquely enabled by photo-affinity labeling (PAL) coupled with chemical enrichment and unbiased analysis by mass spectrometry (MS). MS-based binding site hotspot maps provide structural resolution of interaction sites in conjunction with identification of target proteins. However, binding site hotspot mapping has been confined to relatively simple small molecules to date; extension to more complex compounds would enable the structural definition of new binding modes in the proteome. Here, we extend PAL and MS methods to derive a binding site hotspot map for the immunosuppressant rapamycin, a complex macrocyclic natural product that forms a ternary complex with the proteins FKBP12 and FRB. Photo-rapamycin was developed as a diazirine-based PAL probe for rapamycin, and the FKBP12–photo-rapamycin–FRB ternary complex formed readily in vitro. Photo-irradiation, digestion, and MS analysis of the ternary complex revealed a McLafferty rearrangement product of photo-rapamycin conjugated to specific surfaces on FKBP12 and FRB. Molecular modeling of the ternary complex based on the binding site map revealed a 5.0 Å minimum distance constraint between the conjugated residues and the diazirine carbon. Molecular dynamics further predicted a 9.0 Å labeling radius for the diazirine upon photo-activation that may be useful in the interpretation of binding site measurements from PAL more broadly. Thus, in characterizing the ternary complex of photo-rapamycin by MS, we applied binding site hotspot mapping to a macrocyclic natural product and extracted a precise structural measurement for interpretation of PAL products that may enable the discovery of new ligand space in the “undruggable” proteome.

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Analysis of pyrenoid proteome in the diatom Phaeodactylum tricornutum

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