Pea PSII-LHCII supercomplexes form pairs by making connections across the stromal gap

In higher plant thylakoids, the heterogeneous distribution of photosynthetic protein complexes is a
determinant for the formation of grana, stacks of membrane discs that are densely populated with
Photosystem II (PSII) and its light harvesting complex (LHCII). PSII associates with LHCII to form the
PSII-LHCII supercomplex, a crucial component for solar energy conversion. Here, we report a biochemical,
structural and functional characterization of pairs of PSII-LHCII supercomplexes, which were isolated under
physiologically-relevant cation concentrations. Using single-particle cryo-electron microscopy, we
determined the three-dimensional structure of paired C2S2M PSII-LHCII supercomplexes at 14 Å resolution.
The two supercomplexes interact on their stromal sides through a specific overlap between apposing LHCII
trimers and via physical connections that span the stromal gap, one of which is likely formed by interactions
between the N-terminal loops of two Lhcb4 monomeric LHCII subunits. Fast chlorophyll fluorescence
induction analysis showed that paired PSII-LHCII supercomplexes are energetically coupled. Molecular
dynamics simulations revealed that additional flexible physical connections may form between the apposing
LHCII trimers of paired PSII-LHCII supercomplexes in appressed thylakoid membranes. Our findings
provide new insights into how interactions between pairs of PSII-LHCII supercomplexes can link adjacent
thylakoids to mediate the stacking of grana membranes.