Rocking The Photosynthetic Boat: Peridinin-chlorophyll-protein
Created by Hans Verkerke
Peridinin-chlorophyll-protein (1PPR) from the photosynthetic dinoflagellate Amphidinium carterae is a soluble light-harvesting antenna complex. Composed almost entirely of alpha helices and shaped like a boat, the structure of peridinin-chlorophyll-protein (PCP) relates directly to its function. Antenna complexes with similar function but unrelated structure occur in both higher plants and lineages of photosynthetic bacteria, indicating their functional importance in autotrophy (1). Peridinin-chlorophyll-protein (PCP) is unique among these complexes. It predominately binds the carotenoid pigment peridinin rather than chlorophyll, the primary pigment molecule of terrestrial photosynthetic lineages (2).
The structure of the PCP monomer approximates a hydrophobic scaffold, in which peridinin molecules are oriented to maximize excitation energy transfer to a central chlorophyll alpha molecule (8). Oligomerization is context dependent and yields a stable trimer (subunits M-O). Isoforms of PCP are encoded by a cluster of adjacent genes. Under high salt conditions, a structurally distinct isoform of PCP is expressed (PDB ID=2C9E). BLAST sequence and DALI structural comparisons indicated extensive similarities between high and low salt isoforms of PCP (12). Nonetheless, spectral studies reveal important differences in the number and orientation of bound peridinin molecules. Presence of a context dependent isoform of PCP with conserved structural features highlights the importance of this antenna complex for the light harvesting capacity of dinoflagellates (7).
Approximately 40% of photosynthesis on earth occurs in aquatic environments, where autotrophic organisms form the base of the vast majority of food chains (1). Dinoflagellates are notable among aquatic photosynthetic eukaryotes for using predominately carotenoids over chlorophylls for light harvesting (2). This adaptation allows dinoflagellate lineages to use a larger range of photonic wavelengths to facilitate photosynthesis. PCP and the unrelated chloroplast membrane light-harvesting complex (LHC) mediate excitation energy transfer between associated pigments and reaction centers in the thylakoid membrane, driving photosynthesis (6).
The PCP complex appears only in dinoflagellates, where its function has not been fully elucidated. Much like higher plants, dinoflagellates possess two photososystems with a thylakoid membrane associated light-harvesting complex that transfers excitation energy into reaction centers (1). Whereas higher plants typically use chlorophylls as pigments in both peripheral and integral light harvesting processes, dinoflagellates and a number of algal lineages have evolved to use carotenoids. Observations in A. carterae suggest that PCP serves as the primary reservoir for the carotenoid pigment, peridinin (2). Approximately 50% of the peridinin available in cells resides in this light-harvesting molecule (8).
Carotenoids are natural pigment molecules that absorb the highest energy cross-section of sunlight. In addition to their function as excitation energy donors in the photosystems of marine autotrophs, carotenoids protect photosynthetic machinery from high-energy oxidizing radiation (2). Structurally, carotenoids consist of symmetric or asymmetric head- groups linked by chains of conjugated isoprenoid units (8). The use of many classes of carotenoids has evolved among photosynthetic organisms. But peridinin occurs only in dinoflagellate lineages (2). Peridinin is an asymmetric carotenoid with four main functionalities: 1) A terminal epoxycyclohexane ring 2) a polyene chain 3) a furanic ring 4) and a terminal cyclohexane ring. It absorbs electromagnetic radiation in the cross section between 470 nm and 550 nm (8). In the PCP antenna complex, excited peridinin molecules are oriented to maximize Forster dipole energy transfer from the S1 energy level to a central chlorophyll α molecule (9).
PCP has been extensively studied in vitro. The structural model of PCP, obtained by X-ray protein crystallography, revealed the relative orientations and conformations of bound peridinin and chlorophyll molecules within the holoprotein (8). Follow up studies on reconstituted forms of the protein have provided insight into the mechanisms of excitation energy transfer between pigment molecules (4). The PCP monomeric apoprotein has a molecular weight of 9.7 kDa and its isoelectric point (pI) is 5.63. The trimeric holoprotein has an estimated molecular weight of 38.1 kDa (8). The seminal crystallographic study by Hofmann et al. sparked numerous investigations into the structural features of this unique complex and its various isoforms. These structural insights have begun to provide clues into the dynamic role that PCPs play in dinoflagellate photosynthesis.
Monomeric PCP has two domains, both alpha helical scaffolds, supporting clusters of light harvesting pigment molecules (7). In A. carterae, these associated domains form either from two 16 kDa monomers or from a 32kDa long form polypeptide, which likely arose from a fusion of tandem repeats in the gene that encoded an ancestral form of PCP. In each subunit, The N- and C- terminal domains of long form PCP (LF-PCP) share approximately 56% sequence homology and a twofold pseudosymmetry axis. The 8 alpha helices in each domain form an open “jellyroll” configuration, the tip of which is formed by a β-hairpin (8).
Upon folding by an unknown mechanism, the N- and C- terminal domains each form boat-like structures encasing a hydrophobic space of approximately 23 nm3 within which are arranged 8 peridinin molecules around 2 chlorophyll alpha molecules. Alpha helices that form the bow of the N-terminal domain align to form hydrophobic interactions with the C-terminal alpha helices of the second domain (7)
Openings near the bow of the N-terminal domain and the stern of the C-terminal domain are filled with the hydrophilic epoxycyclohexane rings of peridinin and the head groups of digalactosyl diacyl glycerol (DGDG). DGDG, a lipid, binds to chlorophyll molecules during PCP folding (8). A spectral study of reconstituted PCP formed by two N-terminal domain monomers revealed ligand orientation in the hydrophobic space (10). Re-naturation studies have shown that DGDG significantly improves yields of the native protein (5).
These subunits assemble into a stable non-crystallographic trimer (diameter of 10nm and thickness of 4nm under physiological conditions. The complex consists of 24 α-helices: 3 structurally identical subunits each consisting of 8 alpha helices. Trimerization is mediated by hydrophobic interactions between the helices of the stern and deck regions of either 16 kDa or 32 kDa monomers (8). In addition, a peridinin molecule and its symmetry mates along the trimer axis stabilize the complex by hydrogen bonding with an associated water molecule (7). At low pH and low Mg2+ concentration the monomer-trimer equilibrium has been observed to shift in favor of the monomeric state. When saturated with light, the photosystems of higher plants pump protons into the thylakoid lumen, lowering the pH. Charge is balanced by the flow of magnesium ions out of the lumen and into the stromal region (1). This process is known to modulate the equilibrium between trimer and monomer states of plant light harvesting systems. Analogous regulation presumably occurs in A. carterae (7).
Integral to the structure of chlorophyll alpha is a tetrapyrrole ring bound to Mg2+, which conjugates to a water molecule. It is through interaction with this ring that PCP orients the chlorophyll molecule to the four peripheral peridinin molecules. The imidazole rings of two conserved histidine residues (His-66 and His-229) protrude within van der Waals distance of the tetrapyrrole structure in each domain to hydrogen bond with a magnesium-conjugated water molecule (8). A number of residues in the non-polar interior space protrude in close proximity to the non-polar regions of the peridinin molecules. These residues include Ile-48, Ile-44, Ile-70, and Ala-63 (10). The overall effect of creating this hydrophobic interior is to decrease the entropically unfavorable ordering of solvent molecules.
Site directed mutagenesis was used to study the effects of single amino acid changes in the sequence of an N-terminal refolded PCP complex, dubbed RFPCP by researchers (4). Long-form reconstituted systems exist, but oligomers formed by the truncated RFPCP provide a simplified protein environment for the study of important residues in the pigment reservoir (7). Mutations that do not significantly affect the structure of PCP appear not to alter its function—absorption by peridinin and excitation transfer to chlorophyll (8). However, exceptions to this observation exist. Of particular interest is the mutation N89L (PDB=3IIU), which displays a spectral shift of 25nm into the blue region of the electromagnetic spectrum (12). This asparagine residue appears to be responsible for holding peridinin-614 (per-614) in its orientation closest to the central chlorophyll molecule. This is accomplished through polar interactions of the amide functionality with the conjugated π-system of the carotenoid (3). The mutation does not affect the efficiency of energy transfer from the other 3 peridinin molecules to the chlorophyll. This observation suggests a unique role for per-614 as a photo-protective species. Photo-protection is typically accomplished by quenching of the triplet state in chlorophyll, ultimately preventing the formation of reactive oxygen species. Calculations suggest that per-614 is oriented by Asn-89 at the proper distance for triplet quenching, highlighting the importance of this residue in PCP function (12).
PCP complexes share similar functions with a number of conserved light harvesting antennae like LHCII of higher plants and LH2 of photosynthetic bacteria. These complexes all mediate energy transfer to reaction centers while serving important photo-protective roles. But PCP does not share significant sequence homology with any known proteins. It is also structurally distinct from these complexes (7).
The high salt form of peridinin-chlorophyll-protein (2C9E) from A. carterae is a member of the PCP gene cluster preferentially expressed under saline conditions (7). The results of DALI (Z=30.7 rmsd=2.0) and protein Blast (E=3e-26) revealed that the high salt (HSPCP) and low salt forms of PCP have similar structures and nearly identical amino acid sequences. At the levels of sequence and basic structure, PCP and HSPCP are quite similar; yet important differences exist.
HSPCP has a higher percentage of basic residues causing it to elute more slowly from a cation exchange column. The absorption spectrum reveals that only six peridinin molecules are bound in monomeric HSPCP. The low salt PCP monomer contains eight peridinin molecules. Per-612 and Per-622 are missing from the HSPCP (3). In low salt PCP, spectral analysis suggests that Per-612 may transfer its excitation energy only to other peridinin molecules and never directly to the central chlorophyll. This observation may explain the preservation of nearly 100% transfer efficiency in the high salt form where the molecule is missing. The primary differences between the two proteins appear to lie in different folding mechanisms. Refolding experiments have largely failed with the limited supply of HSPCP available for study. Researchers have proposed that HSPCP may require chaperone proteins to facilitate folding (7).