Xiaowei Pan

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.

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.

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.

Structural insights into the catalytic mechanism of aldehyde-deformylating oxygenases.

The fatty alk(a/e)ne biosynthesis pathway found in cyanobacteria gained tremendous attention in recent years as a promising alternative approach for biofuel production. Cyanobacterial aldehyde-deformylating oxygenase (cADO), which catalyzes the conversion of Cn fatty aldehyde to its corresponding Cn-1 alk(a/e)ne, is a key enzyme in that pathway. Due to its low activity, alk(a/e)ne production by cADO is an inefficient process. Previous biochemical and structural investigations of cADO have provided some information on its catalytic reaction. However, the details of its catalytic processes remain unclear. Here we report five crystal structures of cADO from the Synechococcus elongates strain PCC7942 in both its iron-free and iron-bound forms, representing different states during its catalytic process. Structural comparisons and functional enzyme assays indicate that Glu144, one of the iron-coordinating residues, plays a vital role in the catalytic reaction of cADO. Moreover, the helix where Glu144 resides exhibits two distinct conformations that correlates with the different binding states of the di-iron center in cADO structures. Therefore, our results provide a structural explanation for the highly labile feature of cADO di-iron center, which we proposed to be related to its low enzymatic activity. On the basis of our structural and biochemical data, a possible catalytic process of cADO was proposed, which could aid the design of cADO with improved activity.

Crystal Structure Analysis of Extrinsic PsbP Protein of Photosystem II Reveals a Manganese- Induced Conformational Change

The Mn cluster (Mn4CaO5) on the thylakoid luminal side of photosystem II (PSII) catalyzes the photosynthetic oxygen-evolving reaction, an essential process for life on Earth. In higher plants and green algae, the Mn cluster is surrounded by the membrane-extrinsic proteins PsbO (33 kDa), PsbP (23 kDa), and PsbQ (17 kDa) (Murata and Miyao, 1985). They shield the Mn cluster, protecting and stabilizing it from attack by exogenous reductants. PsbP plays an indispensable role in PSII function, and is necessary for the retention of Ca2+ and Cl– (Ghanotakis et al., 1984), the assembly of PSII complex (Yi et al., 2007), and the maintenance of normal thylakoid architecture (Yi et al., 2009).

Crystal structure studies on sulfur oxygenase reductase from Acidianus tengchongensis

Sulfur oxygenase reductase (SOR) simultaneously catalyzes oxidation and reduction of elemental sulfur to produce sulfite, thiosulfate, and sulfide in the presence of molecular oxygen. In this study, crystal structures of wild type and mutants of SOR from Acidianus tengchongensis (SOR-AT) in two different crystal forms were determined and it was observed that 24 identical SOR monomers form a hollow sphere. Within the icosatetramer sphere, the tetramer and trimer channels were proposed as the paths for the substrate and products, respectively. Moreover, a comparison of SOR-AT with SOR-AA (SOR from Acidianus ambivalens) structures showed that significant differences existed at the active site. Firstly, Cys31 is not persulfurated in SOR-AT structures. Secondly, the iron atom is five-coordinated rather than six-coordinated, since one of the water molecules ligated to the iron atom in the SOR-AA structure is lost. Consequently, the binding sites of substrates and a hypothetical catalytic process of SOR were proposed.

Conformational Changes of rBTI from Buckwheat upon Binding to Trypsin: Implications for the Role of the P8′ Residue in the Potato Inhibitor I Family

BWI-1 (buckwheat trypsin inhibitor), a member of the potato inhibitor I family, suppresses the growth of T-acute lymphoblastic leukemia cells and induces apoptosis in human solid tumor cell lines. Here, we report the crystal structure of rBTI (recombinant buckwheat trypsin inhibitor), a recombinant protein of BWI-1, at 1.84 Å resolution and the structure of rBTI in complex with bovine trypsin at 2.26 Å resolution. A conformational change of Trp53 at the P8′ position in rBTI was observed upon its binding to trypsin, which is not seen in other members of the potato inhibitor I family reported previously. The role of the P8′ residue in the potato inhibitor I family was examined by measuring the association and dissociation rates of four rBTI mutants with different substitutions at the P2 and P8′ positions when binding to trypsin. One of the mutants, P44T, was found to be a much stronger inhibitor than wild-type rBTI, with a picomolar (pM) dissociation constant. Our results could provide valuable insights for designing a new rBTI-based antitumor drug in the future.

Crystal structures of Pseudomonas syringae pv. tomato DC3000 quinone oxidoreductase and its complex with NADPH.

Zeta-crystallin-like quinone oxidoreductase is NAD(P)H-dependent and catalyzes one-electron reduction of certain quinones to generate semiquinone. Here we present the crystal structures of zeta-crystallin-like quinone oxidoreductase from Pseudomonas syringae pv. tomato DC3000 (PtoQOR) and its complexes with NADPH determined at 2.4 and 2.01A resolutions, respectively. PtoQOR forms as a homologous dimer, each monomer containing two domains. In the structure of the PtoQOR-NADPH complex, NADPH locates in the groove between the two domains. NADPH binding causes obvious conformational changes in the structure of PtoQOR. The putative substrate-binding site of PtoQOR is wider than that of Escherichia coli and Thermus thermophilus HB8. Activity assays show that PtoQOR has weak 1,4-benzoquinone catalytic activity, and very strong reduction activity towards large substrates such as 9,10-phenanthrenequinone. We propose a model to explain the conformational changes which take place during reduction reactions catalyzed by PtoQOR.

Spectroscopic study of the light-harvesting CP29 antenna complex of photosystem II--part I.

Recent structural data revealed that the CP29 protein of higher plant photosystem II (PSII) contains 13 chlorophylls (Chls) per complex (Pan et al. Nat. Struct. Mol. Biol. 2011, 18, 309), i.e., five Chls more than in the predicted CP29 homology-based structure model (Bassi et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 10056). This lack of consensus presents a constraint on the interpretation of CP29 optical spectra and their underlying electronic structure. To address this problem, we present new low-temperature (5 K) absorption, fluorescence, and hole-burned (HB) spectra for CP29 proteins from spinach, which are compared with the previously reported data. We focus on excitation energy transfer (EET) and the nature of the lowest-energy state(s). We argue that CP29 proteins previously studied by HB spectroscopy lacked at least one Chl a molecule (i.e., a615 or a611), which along with Chl a612 contribute to the lowest energy state in more intact CP29, and one Chl b (most likely b607). This is why the low-energy state and fluorescence maxima reported by Pieper et al. (Photochem. Photobiol.2000, 71, 574) were blue-shifted by ~1 nm, the low-energy state appeared to be highly localized on a single Chl a molecule, and the position of the low-energy state was independent of burning fluence. In contrast, the position of the nonresonant HB spectrum shifts blue with increasing fluence in intact CP29, as this state is strongly contributed to by several pigments (i.e., a611, a612, a615, and a610). Zero-phonon hole widths obtained for the Chl b band at 638.5 nm (5 K) revealed two independent Chl b → Chl a EET times, i.e., 4 ± 0.5 and 0.4 ± 0.1 ps. The latter value is a factor of 2 faster than previously observed by HB spectroscopy and very similar to the one observed by Gradinaru et al. (J. Phys. Chem. B 2000, 104, 9330) in pump-probe experiments. EET time from 650 nm Chl b → Chl a and downward EET from Chl(s) a state(s) at 665 nm occurs in 4.9 ± 0.7 ps. These findings provide important constraints for excitonic calculations that are discussed in the accompanying paper (part II, DOI 10.1021/jp4004278 ).

Structure of the catalytic domain of a state transition kinase homolog from Micromonas algae

Under natural environments, plants and algae have evolved various photosynthetic acclimation mechanisms in response to the constantly changing light conditions. The state transition and long-term response processes in photosynthetic acclimation involve remodeling and composition alteration of thylakoid membrane. A chloroplast protein kinase named Stt7/STN7 has been found to have pivotal roles in both state transition and long-term response. Here we report the crystal structures of the kinase domain of a putative Stt7/STN7 homolog from Micromonas sp. RCC299 (MsStt7d) in the apo form and in complex with various nucleotide substrates. MsStt7d adopts a canonical protein kinase fold and contains all the essential residues at the active site. A novel hairpin motif, found to be a conserved feature of the Stt7/STN7 family and indispensable for the kinase stability, interacts with the activation loop and fixes it in an active conformation. We have also demonstrated that MsStt7d is a dualspecifi city kinase that phosphorylates both Thr and Tyr residues. Moreover, preliminary in vitro data suggest that it might be capable of phosphorylating a consensus N-terminal pentapeptide of light-harvesting proteins Micromonas Lhcp4 and Arabidopsis Lhcb1 directly. The potential peptide/protein substrate binding site is predicted based on the location of a pseudo-substrate contributed by the adjacent molecule within the crystallographic dimer. The structural and biochemical data presented here provide a framework for an improved understanding on the role of Stt7/STN7 in photosynthetic acclimation.