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Oligomeric assemblies of plant biotin carboxylase revealed by cryo-EM and cross-linking

ABSTRACT: Acetyl-CoA carboxylase (ACCase) initiates de novo fatty acid synthesis in plastids, yet no experimental structure has been available for any plastid biotin carboxylase (BC) subunit. Here, we report the first cryo-EM structures of BC from Thlaspi arvense (pennycress), a developing oilseed crop. Purified recombinant BC predominantly formed a dimer, resolved at 2.85 Å, with architecture highly similar to bacterial heteromeric ACCase BCs. Rare tetrameric particles (<1%) yielded an ~8 Å reconstruction revealing a distinct “kissing” interface between A domains that differs from previously described BCCP-induced tetramers. Bioinformatic comparison with bacterial homologs and normal mode analysis identified a highly dynamic B-domain lid whose motions are consistent with nucleotide-dependent opening and closure. Consistently, lysine acetylation mapping showed strong reactivity in the exposed B-domain, supporting intrinsic mobility. Crosslinking mass spectrometry at higher BC concentration captured dimer–dimer interactions that could not be explained by the symmetric cryo-EM tetramer. Crosslink-guided modeling instead supported an asymmetric dimer of dimers with rotated, translated, and tilted orientations that partially restrict lid opening. These results provide the first structural framework for a plastid BC and reveal multiple oligomeric architectures, including an asymmetric assembly that may underlie higher-order organization observed in plant chloroplasts.

INSTRUMENT(S):

ORGANISM(S): Thlaspi Arvense

SUBMITTER: Gabriel Lemes Jorge

LAB HEAD: Steven R. Van Doren

PROVIDER: PXD071146 | Pride | 2026-03-30

REPOSITORIES: Pride

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			Action	DRS
	1_Thermo_ProteomeDiscoverer_5samples_raws.zip	Other
	1_Thermo_ProteomeDiscoverer_5samples_results.zip	Other
	2_Thermo_Proteome_Discoverer_15samples_raws.zip	Other
	2_Thermo_Proteome_Discoverer_15samples_results.zip	Other
	3_Bruker_PEAKS_20_samples_raws.zip	Other

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Project description:Acetylation of amino groups is an important and recurrent protein modification in all eukaryotes. In plants, both lysine and N-terminal acetylation have additional important roles in protein regulation in chloroplasts. However, it is yet unclear how acetylation patterns respond to environmental stresses, and whether lysine or N-terminal modifications similarly contribute to the associated perturbations. A new family of plastid acetyltransferase, called GNATs, was recently discovered, which consists of closely related enzymes featuring both lysine- and N-terminal acetyltransferase activities often on the same polypeptide targets. Here, we take advantage of this unique characteristic of Arabidopsis GNAT2 to obtain a holistic multi-omics acetylation-dependent view during the acclimation of plants to short term changes in light. No substantial difference in transcriptome or adenylate energy charge oscillations were observed between WT and gnat2 knockout mutant lines when they were submitted for two hours to high-light or dark growth conditions. A detailed characterization of the N-terminal acetylome reveals that both its yield and coverage remain unchanged upon different light conditions in both genotypes. Unlike the N-terminal acetylome, the GNAT2-associated lysine acetylome is sensitive to the different light conditions used. In addition, a strong reduction in both types of acetylations on several plastid proteins was observed upon GNAT2 inactivation under all light conditions. Our data suggests that the lysine acetylome marks on proteins more rapidly fluctuate following acclimation to the environmental condition, while N-terminal acetylation changes are associated to longer term responses, like those promoted in the gnat2 background. Taken together, our data revealed unique strategies of plant acclimation to the different applied treatments involving specific PTMs and emphasizes distinct timescale responses of plastid lysine and N-terminal acetylomes to environmental changes.

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Oligomeric assemblies of plant biotin carboxylase revealed by cryo-EM and cross-linking

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