Wenrui Chang

Crystal structure of spinach major light-harvesting complex at 2.72 Å resolution

The major light-harvesting complex of photosystem II (LHC-II) serves as the principal solar energy collector in the photosynthesis of green plants and presumably also functions in photoprotection under high-light conditions. Here we report the first X-ray structure of LHC-II in icosahedral proteoliposome assembly at atomic detail. One asymmetric unit of a large R32 unit cell contains ten LHC-II monomers. The 14 chlorophylls (Chl) in each monomer can be unambiguously distinguished as eight Chla and six Chlb molecules. Assignment of the orientation of the transition dipole moment of each chlorophyll has been achieved. All Chlb are located around the interface between adjacent monomers, and together with Chla they are the basis for efficient light harvesting. Four carotenoid-binding sites per monomer have been observed. The xanthophyll-cycle carotenoid at the monomer–monomer interface may be involved in the non-radiative dissipation of excessive energy, one of the photoprotective strategies that have evolved in plants.

Molecular basis of photoprotection and control of photosynthetic light-harvesting

In order to maximize their use of light energy in photosynthesis, plants have molecules that act as light-harvesting antennae, which collect light quanta and deliver them to the reaction centres, where energy conversion into a chemical form takes place. The functioning of the antenna responds to the extreme changes in the intensity of sunlight encountered in nature. In shade, light is efficiently harvested in photosynthesis. However, in full sunlight, much of the energy absorbed is not needed and there are vitally important switches to specific antenna states, which safely dissipate the excess energy as heat. This is essential for plant survival, because it provides protection against the potential photo-damage of the photosynthetic membrane. But whereas the features that establish high photosynthetic efficiency have been highlighted, almost nothing is known about the molecular nature of the dissipative states. Recently, the atomic structure of the major plant light-harvesting antenna protein, LHCII, has been determined by X-ray crystallography. Here we demonstrate that this is the structure of a dissipative state of LHCII. We present a spectroscopic analysis of this crystal form, and identify the specific changes in configuration of its pigment population that give LHCII the intrinsic capability to regulate energy flow. This provides a molecular basis for understanding the control of photosynthetic light-harvesting.

Structural insights into energy regulation of light-harvesting complex CP29 from spinach

CP29, one of the minor light-harvesting complexes of higher-plant photosystem II, absorbs and transfers solar energy for photosynthesis and also has important roles in photoprotection. We have solved the crystal structure of spinach CP29 at 2.80-Å resolution. Each CP29 monomer contains 13 chlorophyll and 3 carotenoid molecules, which differs considerably from the major light-harvesting complex LHCII and the previously proposed CP29 model. The 13 chlorophyll-binding sites are assigned as eight chlorophyll a sites, four chlorophyll b and one putative mixed site occupied by both chlorophylls a and b. Based on the present X-ray structure, an integrated pigment network in CP29 is constructed. Two special clusters of pigment molecules, namely a615–a611–a612–Lut and Vio(Zea)–a603–a609, have been identified and might function as potential energy-quenching centers and as the exit or entrance in energy-transfer pathways.

Structure of spinach photosystem II–LHCII supercomplex at 3.2 Å resolution

During photosynthesis, the plant photosystem II core complex receives excitation energy from the peripheral light-harvesting complex II (LHCII). The pathways along which excitation energy is transferred between them, and their assembly mechanisms, remain to be deciphered through high-resolution structural studies. Here we report the structure of a 1.1-megadalton spinach photosystem II-LHCII supercomplex solved at 3.2 Å resolution through single-particle cryo-electron microscopy. The structure reveals a homodimeric supramolecular system in which each monomer contains 25 protein subunits, 105 chlorophylls, 28 carotenoids and other cofactors. Three extrinsic subunits (PsbO, PsbP and PsbQ), which are essential for optimal oxygen-evolving activity of photosystem II, form a triangular crown that shields the Mn4CaO5-binding domains of CP43 and D1. One major trimeric and two minor monomeric LHCIIs associate with each core-complex monomer, and the antenna-core interactions are reinforced by three small intrinsic subunits (PsbW, PsbH and PsbZ). By analysing the closely connected interfacial chlorophylls, we have obtained detailed insights into the energy-transfer pathways between the antenna and core complexes.

Purification, characterization and crystallization of a group of earthworm fibrinolytic enzymes from Eisenia fetida

Seven fibrinolytic enzymes were purified from the earthworm Eisenia fetida. The molecular weights of the enzymes were 24 663, 29 516, 29 690, 24 201, 24 170, 23 028 and 29 595, and the respective isoelectric points were 3.46, 3.5, 3.5, 3.68, 3.62, 3.94 and 3.46. All the proteases showed different fibrinolytic activity on fibrin plates. Studies on substrate specificity and inhibition indicated that they belonged to different types of serine proteases. N-Terminal sequencing indicated their high homology to those from the earthworm Lumbricus rubellus. All the enzymes have been crystallized.

Crystal Structure of Earthworm Fibrinolytic Enzyme Component A: Revealing the Structural Determinants of its Dual Fibrinolytic Activity

Earthworm fibrinolytic enzyme component A (EFEa) from Eisenia fetida is a strong fibrinolytic enzyme that not only directly degrades fibrin, but also activates plasminogen. Proteolytic assays further revealed that it cleaved behind various P1 residue types. The crystal structure of EFEa was determined using the MIR method and refined to 2.3A resolution. The enzyme, showing the overall polypeptide fold of chymotrypsin-like serine proteases, possesses essential S1 specificity determinants characteristic of elastase. However, the beta strand at the west rim of the S1 specificity pocket is significantly elongated by a unique four-residue insertion (Ser-Ser-Gly-Leu) after Val217, which not only provides additional substrate hydrogen binding sites for distal P residues, but also causes extension of the S1 pocket at the south rim. The S2 subsite of the enzyme was partially occluded by the bulky side-chain of residue Tyr99. Structure-based inhibitor modeling demonstrated that EFEas S1 specificity pocket was preferable for elastase-specific small hydrophobic P1 residues, while its accommodation of long and/or bulky P1 residues was also feasible if enhanced binding of the substrate and induced fit of the S1 pocket were achieved. EFEa is thereby endowed with relatively broad substrate specificity, including the dual fibrinolysis. The presence of Tyr99 at the S2 subsite indicates a preference for P2-Gly, while an induced fit of Tyr99 was also suggested for accommodation of bigger P2 residues. This structure is the first reported for an earthworm fibrinolytic enzyme component and serine protease originating from annelid worms.

Crystal structures of the PsbS protein essential for photoprotection in plants

The photosystem II protein PsbS has an essential role in qE-type nonphotochemical quenching, which protects plants from photodamage under excess light conditions. qE is initiated by activation of PsbS by low pH, but the mechanism of PsbS action remains elusive. Here we report the low-pH crystal structures of PsbS from spinach in its free form and in complex with the qE inhibitor N,N′-dicyclohexylcarbodiimide (DCCD), revealing that PsbS adopts a unique folding pattern, and, unlike other members of the light-harvesting-complex superfamily, it is a noncanonical pigment-binding protein. Structural and biochemical evidence shows that both active and inactive PsbS form homodimers in the thylakoid membranes, and DCCD binding disrupts the lumenal intermolecular hydrogen bonds of the active PsbS dimer. Activation of PsbS by low pH during qE may involve a conformational change associated with altered lumenal intermolecular interactions of the PsbS dimer.

Structure of C-phycocyanin from Spirulina platensis at 2.2 Å resolution: a novel monoclinic crystal form for phycobiliproteins in phycobilisomes

The crystal structure of C-phycocyanin from the cyanobacterium S. platensis has been determined at 2.2 A resolution. The crystals belong to the monoclinic crystal form, which has not been previously reported for phycobiliprotein structures. The structure was solved using the molecular-replacement method with a final R value of 18.9% (R(free) = 23.7%) after model building and refinement. In the crystals used for the study, the C-phycocyanin hexamers formed by face-to-face association of two trimers are arranged in layers rather than in columns. Three different kinds of packing between adjacent hexamers in the layer were compared. The tight packing of two adjacent hexamers formed by four trimers in the asymmetric unit brings beta155 PCB chromophores close together, so it is possible that lateral energy transfer takes place through the beta155-beta155 route.

Structural Basis for Thermostability of beta-Glycosidase from the Thermophilic Eubacterium Thermus nonproteolyticus HG102.

The three-dimensional structure of a thermostable beta-glycosidase (Gly(Tn)) from the thermophilic eubacterium Thermus nonproteolyticus HG102 was determined at a resolution of 2.4 A. The core of the structure adopts the (betaalpha)(8) barrel fold. The sequence alignments and the positions of the two Glu residues in the active center indicate that Gly(Tn) belongs to the glycosyl hydrolases of retaining family 1. We have analyzed the structural features of Gly(Tn) related to the thermostability and compared its structure with those of other mesophilic glycosidases from plants, eubacteria, and hyperthermophilic enzymes from archaea. Several possible features contributing to the thermostability of Gly(Tn) were elucidated.

Architecture and function of plant light-harvesting complexes II

The antenna system associated with plant photosystem II (PSII) comprises a series of light-harvesting complexes II (LHCIIs) which are supramolecular assemblies of chlorophylls, carotenoids, lipids and integral membrane proteins. These complexes not only function in capturing and transmitting light energy, but also have pivotal roles in photoprotection under high-light conditions through a mechanism known as non-photochemical quenching process. Among them, the most abundant major species (majLHCII) is located at the periphery of PSII and forms homo/hetero-trimers. Besides, three minor species, named CP29, CP26 and CP24, are adjacent to the PSII core, exist in monomeric form and bridge the majLHCII trimers with the core complex. Structural studies on majLHCII and CP29 have revealed the overall architecture of plant LHC family, the binding sites of pigment molecules and the distribution pattern of chromophores in three-dimensional space. The high-resolution structural data of LHCIIs serve as fundamental bases for an improved understanding on the mechanisms of light harvesting, energy transfer and photoprotection processes in plants.