How paired PSII-LHCII supercomplexes mediate the stacking of plant thylakoid membranes unveiled by integrative structural mass-spectrometry

ABSTRACT: Photosynthesis drives all life on Earth by exploiting solar energy to split water molecules through the Photosystem (PS) II enzyme. In plant thylakoid membranes, PSII binds a modular set of light harvesting complexes (LHCII) to form different types of PSII-LHCII supercomplex (PSII-LHCIIsc). Plant PSII-LHCIIsc are localized in the stacked region of the thylakoid membranes called grana, from which they can be isolated in paired conformations of type (C2S2M2)x2 and (C2S2M)x2, with the two supercomplexes facing each other at their stromal surface. Although the atomic structure of PSII-LHCIIsc has recently been solved, there is still a lack of knowledge on their mutual interactions when facing each other within apposing thylakoid membranes, as well as their structural dynamics in response to light variations. The major challenges in the structural determination of the stromal interactions are posed not only by the dynamic nature of these over 2-megadalton assemblies, but also by the heterogeneity of the LHCII subunits and the high flexibility of their stromally exposed N-terminal loops. Here, we explored the potential of combining top-down mass spectrometry (TD-MS) and crosslinking mass spectrometry (XL-MS) to peek through the keyhole of the tight stromal gap between two facing supercomplexes, unveiling so far hidden structural details. A first goal of experiments was to identify the distinct sequence variants and proteoforms involved in PSII-LHCIIsc structural dynamics in response to light modulation. To investigate their structural interactions, we treated paired PSII-LHCIIsc isolated from the three light conditions with two complementary chemical cross-linkers, targeting different residues and producing partially overlapping distance restraints. Most interactions detected “in-vitro” on the isolated paired supercomplexes were further supported by XL-MS results obtained “in-situ” on the corresponding thylakoid membranes.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive HF

ORGANISM(S): Pisum Sativum (garden Pea)

TISSUE(S): Photosynthetic Cell, Leaf

SUBMITTER: Pascal Albanese  

LAB HEAD: Richard Scheltema

PROVIDER: PXD017382 | Pride | 2020-03-19

REPOSITORIES: Pride