Biology subjects

Hunter, C. N.

Publications and source records attributed to Hunter, C. N..

7 recordsLinked to original sources

bioRxiv2026

Cryo-EM structures of Cereibacter sphaeroides ferrochelatase reveal an oligomeric state that occludes the substrate-binding site

Protoporphyrin IX (PPIX) is the last shared intermediate of heme and (bacterio)chlorophyll ((B)Chl) biosynthesis. Insertion of iron into PPIX by the PPIX ferrochelatase (FeCh) PpfC produces heme, whereas magnesium insertion by magnesium chelatase (MgCh) initiates (B)Chl synthesis. Chlorophototrophic organisms must regulate this biosynthetic node to ensure the correct flux of PPIX to each of these crucial tetrapyrrole products, presenting a particularly acute problem for the facultative bacterium Cereibacter (previously Rhodobacter) sphaeroides, which can switch between chemotrophic (respiratory) and phototrophic (photosynthetic) growth modes. To elucidate the structural basis for the catalytic and regulatory functions of C. sphaeroides FeCh (PpfCCs), we used cryogenic electron microscopy to determine its structure, to our knowledge the first such information for a Gram-negative or phototrophic organism. We determined the 2.6 [A] structure of an active site mutant of PpfCCs, which forms a dimer with a monomer-to-monomer interface that coordinates two hemes. We also determined a 2.5 [A] structure of the wild-type enzyme, in which two dimers interact in a homotetrameric arrangement that occludes PPIX binding at the active site of each monomer. We propose that the PpfCCs dimer likely docks onto the inner membrane in a manner analogous to the human enzyme, whereas the tetramer represents a transiently inactive state whereby the hydrophobic lips and active site are both occluded. These novel oligomerization states of FeCh may have evolved to influence the flux of tetrapyrroles through the heme/BChl branchpoint in purple bacteria.

biochemistry↗

bioRxiv2026

A 13-subunit c-ring in the Chlamydomonas chloroplast ATP synthase lowers the H⁺/ATP cost of carbon fixation

The chloroplast F1Fo ATP synthase is a rotary motor that converts the light-driven proton-motive force into the chemical energy of ATP. The number of c-subunits in its rotor fixes the number of protons translocated per ATP formed, a fundamental parameter of bioenergetic systems. The reference spinach enzyme possesses fourteen c-subunits and a H+/ATP ratio of 4.67. Green algae additionally operate a carbon-concentrating mechanism that sustains CO2 fixation in water at a substantial cost in ATP, yet the structure of the algal motor, and whether its bioenergetic parameters differ from those of vascular plants, remains unresolved. Here, a 2.2 [A] structure of the ATP synthase of Chlamydomonas reinhardtii reveals that the enzyme carries a thirteen-membered c-ring, the first departure from c14 in a chloroplast, and with a lower predicted H+/ATP ratio of 4.33. Ordered waters trace a Grotthuss proton relay through the membrane, where an insulating triad separates the proton loading and unloading sites and couples flux to rotation. A single substitution in the redox switching {gamma}-subunit abolishes the contact with the catalytic {beta}-subunit that idles the enzyme in darkness in vascular plants. These unique features of the algal ATP synthase lower the H+/ATP cost of carbon fixation in the light and facilitate acetate metabolism in the dark.

biochemistry↗

bioRxiv2026

Structure of chlorophyll synthase in complex with the LHC-like protein HliD

Chlorophyll is the central cofactor in oxygenic photosynthesis, responsible for both light capture in antenna complexes and light-driven charge separation in photosystems. The tetraprenyl tail of chlorophyll is attached to the chlorophyllide macrocycle by the transmembrane enzyme chlorophyll synthase (ChlG). In the cyanobacterium Synechocystis sp. PCC 6803, ChlG forms a complex with the LHC-like high-light-inducible protein HliD. To understand the substrate specificity and catalytic mechanism of ChlG, and how it is photoprotected by HliD, we determined the structure of a ChlG2HliD2 complex in both substrate-free and geranylgeranyl pyrophosphate (GGPP)-bound states using cryogenic electron microscopy (cryo-EM). A homodimer of HliD, which binds four chlorophylls and two zeaxanthin molecules in a quenched state, is flanked by two ChlG monomers. AlphaFold modelling placed chlorophyllide adjacent to the structurally resolved GGPP bound to the active site of ChlG. Cryo-EM data, site-directed mutagenesis and molecular dynamics were used to formulate a molecular mechanism for ChlG catalysis. The structure of the ChlG2HliD2 complex shows how Hlips bind to the synthase, reveals the arrangements of carotenoids and chlorophylls that mediate energy dissipation, and sheds light on the evolution of eukaryotic LHC antennae from their cyanobacterial ancestors.

plant biology↗

bioRxiv2024

Sulfoquinovosyl diacylglycerol is required for dimerization of the Rhodobacter sphaeroides RC-LH1 core complex

The reaction centre-light harvesting 1 (RC-LH1) core complex is indispensable for anoxygenic photosynthesis. In the purple bacterium Rhodobacter (Rba.) sphaeroides RC-LH1 is produced both as a monomer in which 14 LH1 subunits form a crescent-shaped antenna around one RC, and as a dimer, where 28 LH1 subunits form an S-shaped antenna surrounding two RCs. The PufX polypeptide augments the five RC and LH subunits, and in addition to providing an interface for dimerization, PufX also prevents LH1 ring closure, introducing a channel for quinone exchange that is essential for photoheterotrophic growth. Structures of Rba. sphaeroides RC-LH1 complexes revealed several new components; protein-Y, which helps to form a quinone channel; protein-Z, of unknown function but which is unique to dimers; and a tightly bound sulfoquinovosyl diacylglycerol (SQDG) lipid that interacts with two PufX arginines. This lipid lies at the dimer interface alongside weak density for a second molecule, previously proposed to be an ornithine lipid. In this work we have generated strains of Rba. sphaeroides lacking protein-Y, protein-Z, SQDG or ornithine lipids to assess the roles of these previously unknown components in the assembly and activity of RC-LH1. We show that whilst the removal of either protein-Y, protein-Z or ornithine lipids has only subtle effects, SQDG is essential for the formation of RC-LH1 dimers but its absence has no functional effect on the monomeric complex.

biochemistry↗

bioRxiv2023

Generalized Biomolecular Modeling and Design with RoseTTAFold All-Atom

Although AlphaFold2 (AF2) and RoseTTAFold (RF) have transformed structural biology by enabling high-accuracy protein structure modeling, they are unable to model covalent modifications or interactions with small molecules and other non-protein molecules that can play key roles in biological function. Here, we describe RoseTTAFold All-Atom (RFAA), a deep network capable of modeling full biological assemblies containing proteins, nucleic acids, small molecules, metals, and covalent modifications given the sequences of the polymers and the atomic bonded geometry of the small molecules and covalent modifications. Following training on structures of full biological assemblies in the Protein Data Bank (PDB), RFAA has comparable protein structure prediction accuracy to AF2, excellent performance in CAMEO for flexible backbone small molecule docking, and reasonable prediction accuracy for protein covalent modifications and assemblies of proteins with multiple nucleic acid chains and small molecules which, to our knowledge, no existing method can model simultaneously. By fine-tuning on diffusive denoising tasks, we develop RFdiffusion All-Atom (RFdiffusionAA), which generates binding pockets by directly building protein structures around small molecules and other non-protein molecules. Starting from random distributions of amino acid residues surrounding target small molecules, we design and experimentally validate proteins that bind the cardiac disease therapeutic digoxigenin, the enzymatic cofactor heme, and optically active bilin molecules with potential for expanding the range of wavelengths captured by photosynthesis. We anticipate that RFAA and RFdiffusionAA will be widely useful for modeling and designing complex biomolecular systems.

biochemistry↗

bioRxiv2022

Enhancing the spectral range of plant and bacterial Light-Harvesting pigment-protein complexes with various synthetic chromophores incorporated into lipid vesicles

The Light-Harvesting (LH) pigment-protein complexes found in photosynthetic organisms have the role of absorbing solar energy with high efficiency and transferring it to reaction centre complexes. LH complexes contain a suite of pigments that each absorb light at specific wavelengths, however, the natural combinations of pigments within any one protein complex do not cover the full range of solar radiation. Here, we provide an in-depth comparison of the relative effectiveness of five different organic "dye" molecules (Texas Red, ATTO, Cy7, Dil, DiR) for enhancing the absorption range of two different LH membrane protein complexes (the major LHCII from plants and LH2 from purple phototrophic bacteria). Proteoliposomes were self-assembled from defined mixtures of lipids, proteins and dye molecules and their optical properties were quantified by absorption and fluorescence spectroscopy. Both lipid-linked dyes and alternative lipophilic dyes were found to be effective excitation energy donors to LH protein complexes, without the need for direct chemical or generic modification of the proteins. The Forster theory parameters (e.g., spectral overlap) were compared between each donor-acceptor combination and found to be good predictors of an effective dye-protein combination. At the highest dye-to-protein ratios tested (over 20:1), the effective absorption strength integrated over the full spectral range was increased to ~180% of its natural level for both LH complexes. Lipophilic dyes could be inserted into pre-formed membranes although their effectiveness was found to depend upon favourable physicochemical interactions. Finally, we demonstrated that these dyes can also be effective at increasing the spectral range of surface-supported models of photosynthetic membranes, using fluorescence microscopy. The results of this work provide insight into the utility of self-assembled lipid membranes and the great flexibility of LH complexes for interacting with different dyes.

biophysics↗

bioRxiv2020

Structure of the light harvesting 2 complex reveals two carotenoid energy transfer pathways in a photosynthetic bacterium

We report the 2.4 [A] resolution structure of the light harvesting 2 complex (LH2) from Marichromatium (Mch.) purpuratum determined by electron cryo-microscopy. The structure contains a heptameric ring that is unique among all known LH2 structures, explaining the unusual spectroscopic properties of this bacterial antenna complex. Two sets of distinct carotenoids are identified in the structure, and a network of energy transfer pathways from the carotenoids to bacteriochlorophyll a molecules is shown. The geometry imposed by the heptameric ring controls the resonant coupling of the long wavelength energy absorption band. Together, these details reveal key aspects of the assembly and oligomeric form of purple bacterial LH2 complexes that were previously inaccessible by any technique. One Sentence SummaryThe structure of a heptameric LH2 antenna complex reveals new energy transfer pathways and the basis for assembling LH rings.

biochemistry↗