Researchers Reveal Chlorophyll Synthase Structure

Researchers in a Nature study revealed the structure of chlorophyll synthase in complex with the LHC-like protein HliD, illuminating how the enzyme binds its substrate.

The study examined the overall architecture of the G-D2-G complex, isolating it with FLAG tags. Using two-dimensional clear-native and SDS–PAGE, the purification yielded the G-D2-G complex alongside a smaller G-D2 complex consisting of a monomer of ChlG bound to two copies of HliD.

To capture the structure with a bound substrate, researchers incubated the isolated complex with 150 µM GGPP before preparing cryo-electron microscopy grids. The resulting structures for the substrate-free apo state and the GGPP-bound state reached resolutions of 3.0–3.4 Å and 3.2–3.6 Å, respectively.

The two halves of the complex are almost identical, each containing one ChlG and one HliD. When incubated with GGPP, a defined extended density appears inside the substrate-binding cavity of each ChlG monomer, surrounded by the $\alpha 2$, $\alpha 4$, and $\alpha 5$ helices. This cavity stays empty in the apo complex.

Conserved residues Lys39, Arg104, and Lys167 create extra electrostatic interactions with the charged pyrophosphate head group of GGPP. Meanwhile, Asp226 on the second entry loop of the cap domain sits roughly 10 Å away—too far to bind GGPP through a coordinated magnesium ion, differing from how conserved counterparts bind substrates in other prenyltransferases.

The tetraprenyl group of GGPP anchors deep inside one of the substrate-binding cavities, surrounded by hydrophobic residues. The loops of the cap domain show the largest structural differences between the apo and GGPP-bound states, indicating that cap flexibility allows GGPP to enter and bind the enzyme.

In cyanobacteria, ChlG binds tightly to HliD, and the absence of HliD compromises the in vivo stability of ChlG. The cryo-EM structure shows that an X-shaped HliD dimer links two ChlG monomers. Aside from connections through zeaxanthin molecules, ChlG monomers interact with the HliD–pigment subcomplex through the carboxy and amino termini of helix $\alpha 8$.

At the lumenal amino terminus of the helix, hydrophobic residues and a possible hydrogen bond between ChlG Gln268 and HliD Trp53 form the interaction. At the cytoplasmic carboxy terminus of ChlG helix $\alpha 8$, electrostatic interactions form between ChlG Asp286 and HliD Arg30.

Further separation of isolated complexes by SDS–PAGE demonstrated that an Asn28Ala HliD mutant yielded a much higher amount of protein than wild-type HliD despite comparable loaded chlorophyll concentrations. This indicates that either a large fraction of the mutant protein binds no pigments or pigments were lost during purification, confirming that chlorophyll a ligated by HliD Asn28 is critical for forming stable chlorophyll and carotenoid-binding sites.